Division of Pediatric Hematology/Oncology, Department of Pediatrics, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA 30322, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Expression Therapeutics, LLC, Tucker, GA 30084, USA.
Mol Ther. 2017 Oct 4;25(10):2372-2382. doi: 10.1016/j.ymthe.2017.07.002. Epub 2017 Jul 8.
Ex vivo gene therapy using lentiviral vectors (LVs) is a proven approach to treat and potentially cure many hematologic disorders and malignancies but remains stymied by cumbersome, cost-prohibitive, and scale-limited production processes that cannot meet the demands of current clinical protocols for widespread clinical utilization. However, limitations in LV manufacture coupled with inefficient transduction protocols requiring significant excess amounts of vector currently limit widespread implementation. Herein, we describe a microfluidic, mass transport-based approach that overcomes the diffusion limitations of current transduction platforms to enhance LV gene transfer kinetics and efficiency. This novel ex vivo LV transduction platform is flexible in design, easy to use, scalable, and compatible with standard cell transduction reagents and LV preparations. Using hematopoietic cell lines, primary human T cells, primary hematopoietic stem and progenitor cells (HSPCs) of both murine (Sca-1) and human (CD34) origin, microfluidic transduction using clinically processed LVs occurs up to 5-fold faster and requires as little as one-twentieth of LV. As an in vivo validation of the microfluidic-based transduction technology, HSPC gene therapy was performed in hemophilia A mice using limiting amounts of LV. Compared to the standard static well-based transduction protocols, only animals transplanted with microfluidic-transduced cells displayed clotting levels restored to normal.
使用慢病毒载体 (LV) 的体外基因治疗是一种已被证实的治疗方法，可以治疗和潜在治愈许多血液系统疾病和恶性肿瘤，但由于生产过程繁琐、成本高、规模有限，无法满足当前广泛临床应用的临床方案的需求，因此仍受到限制。然而，LV 制造的限制以及需要大量多余载体的低效转导方案目前限制了其广泛实施。本文描述了一种基于微流控、质量传递的方法，该方法克服了当前转导平台的扩散限制，从而增强了 LV 基因转移的动力学和效率。这种新型的体外 LV 转导平台设计灵活、易于使用、可扩展，并且与标准细胞转导试剂和 LV 制剂兼容。使用造血细胞系、原代人 T 细胞、来自小鼠 (Sca-1) 和人类 (CD34) 的原代造血干细胞和祖细胞 (HSPC)，使用临床处理的 LV 进行微流控转导的速度快 5 倍，所需的 LV 量仅为原来的 1/20。作为对基于微流控的转导技术的体内验证，使用有限量的 LV 在血友病 A 小鼠中进行了 HSPC 基因治疗。与标准的静态孔板转导方案相比，只有接受微流控转导细胞移植的动物的凝血水平恢复正常。
