1 Department of Surgery, Surgical Bioengineering Laboratory, UC Davis School of Medicine, Research II, University of California, Davis, Sacramento, CA, USA.
2 Department of Burns and Plastic Surgery, The Third Xiangya Hospital of Central South University, Changsha, Hunan, People's Republic of China.
Cell Transplant. 2018 Jan;27(1):130-139. doi: 10.1177/0963689717728937.
Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the factor VIII ( FVIII) gene leading to deficient blood coagulation. The current standard of care is frequent infusions of plasma-derived FVIII or recombinant B-domain-deleted FVIII (BDD-FVIII). While this treatment is effective, many patients eventually develop FVIII inhibitors that limit the effectiveness of the infused FVIII. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Several studies have investigated allogeneic stem cell therapy targeting in utero or postnatal treatment of HA but have not been successful in completely correcting HA. Autologous in utero transplantation of mesenchymal stem cells is promising for treatment of HA due to the naive immune status of the fetal environment as well as its potential to prevent transplant rejection and long-term FVIII inhibitor formation. HA can be diagnosed by chorionic villus sampling performed during the first trimester (10 to 13 wk) of gestation. In this study, we used an established protocol and isolated placenta-derived mesenchymal stromal cells (PMSCs) from first trimester chorionic villus tissue and transduced them with lentiviral vector encoding the BDD-FVIII gene. We show that gene-modified PMSCs maintain their immunophenotype and multipotency, express, and secrete high levels of active FVIII. PMSCs were then transplanted at embryonic day 14.5 (E14.5) into wild-type fetuses from time-mated pregnant mice. Four days after birth, pups were checked for engraftment, and varying levels of expression of human green fluorescent protein were found in the organs tested. This study shows feasibility of the approach to obtain PMSCs from first trimester chorionic villus tissue, genetically modify them with the FVIII gene, and transplant them in utero for cell-mediated gene therapy of HA. Future studies will involve evaluation of long-term engraftment, phenotypic correction in HA mice, and prevention of FVIII inhibitor development by this approach.
血友病 A (HA) 是一种 X 连锁隐性遗传病，由因子 VIII (FVIII) 基因的突变引起，导致血液凝血功能缺陷。目前的标准治疗方法是频繁输注血浆源性 FVIII 或重组 B 结构域缺失 FVIII (BDD-FVIII)。虽然这种治疗方法有效，但许多患者最终会产生 FVIII 抑制剂，从而限制了输注 FVIII 的效果。作为一种单基因疾病，HA 是基因或细胞治疗的理想靶点。已经有几项研究调查了针对 HA 的同种异体干细胞治疗，包括在子宫内或产后进行治疗，但都未能完全纠正 HA。由于胎儿环境的原始免疫状态及其预防移植排斥和长期 FVIII 抑制剂形成的潜力，自体在子宫内移植间充质干细胞治疗 HA 具有很大的前景。HA 可以通过在妊娠早期（10 至 13 周）进行绒毛膜绒毛取样来诊断。在这项研究中，我们使用了已建立的方案，从妊娠早期的绒毛组织中分离胎盘来源的间充质基质细胞 (PMSC)，并将其转导到编码 BDD-FVIII 基因的慢病毒载体中。我们表明，基因修饰的 PMSC 保持其免疫表型和多能性，表达并分泌高水平的活性 FVIII。然后在胚胎第 14.5 天 (E14.5) 将 PMSC 移植到来自同期交配怀孕小鼠的野生型胎儿中。出生后 4 天，检查嵌合体的植入情况，并在测试的器官中发现了不同水平的人绿色荧光蛋白的表达。这项研究表明，从妊娠早期的绒毛组织中获得 PMSC、用 FVIII 基因对其进行基因修饰、并在子宫内移植它们以进行 HA 的细胞介导基因治疗是可行的。未来的研究将包括评估长期植入、HA 小鼠的表型纠正以及通过这种方法预防 FVIII 抑制剂的发展。
