Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis

PMID: 31949009 PMCID: PMC7776247 DOI: 10.3324/haematol.2019.238261
Abstract
Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

PMID: 31949009 PMCID: PMC7776247 DOI: 10.3324/haematol.2019.238261

摘要
同种异体造血干细胞移植是治疗由TCIRG1基因缺陷引起的常染色体隐性成骨不全的首选治疗方法。尽管在预处理方面取得了进展，但由于疾病严重程度和显著的移植相关并发症，仍有相当数量的患者不符合同种异体干细胞移植的条件。我们利用了在TCIRG1缺陷患者外周血中循环频率较高的外周CD34+细胞，作为基因校正细胞自体移植的新细胞来源。详尽的表型分析显示，循环CD34+细胞的细胞组成类似于骨髓，支持其在基因治疗方案中的应用。转录组分析揭示了造血干细胞和祖细胞（HSPCs）表达基因的富集。为了克服TCIRG1患者骨髓采集/ HSPC动员和连续血液采集的局限性，我们应用了基于UM171的HSPCs体外扩增结合慢病毒基因转移技术。TCIRG1缺陷患者的循环CD34+细胞被转导了一种临床优化的慢病毒载体（LV），该载体在甘油酸激酶启动子控制下表达TCIRG1，并在体外扩增。扩增的细胞在体内移植到初级和次级NSG受体后，保持了长期植入能力和多谱系重建造血潜能。此外，当CD34+细胞在体外分化时，基因校正的破骨细胞能够有效地吸收骨骼。总的来说，我们提供了证据，表明循环HSPCs的扩增与基因治疗相结合可以克服成骨不全患者干细胞采集的局限性，从而为未来基于基因的骨髓纤维化引起的骨骼疾病治疗开辟了道路。