Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln - University of Applied Sciences, Chempark Leverkusen E28, Kaiser-Wilhelm-Allee, 51368, Leverkusen, Germany; Institute of Technical Chemistry, Leibniz University Hannover, Callinstraße, 530167, Hannover, Germany.
Research Group Pharmaceutical Biotechnology, Faculty of Applied Natural Sciences, TH Köln - University of Applied Sciences, Chempark Leverkusen E28, Kaiser-Wilhelm-Allee, 51368, Leverkusen, Germany; Research Group Translational Hepatology and Stem Cell Biology, Cluster of Excellence REBIRTH, Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
J Virol Methods. 2021 Nov;297:114243. doi: 10.1016/j.jviromet.2021.114243. Epub 2021 Jul 24.
Retroviral vectors derived from murine leukemia virus (MLV) are amongst the most frequently utilized vectors in gene therapy approaches such as the genetic modification of hematopoietic cells. Currently, vector particles are mostly produced employing adherent viral packaging cell lines (VPCs) rendering the scale up of production laborious, and thus cost-intensive. Here, we describe the rapid establishment of a human suspension 293-F cell line derived ecotropic MLV VPC. Using transposon vector technology, a packaging and envelope expression cassette as well as a transfer vector facilitated the establishment of a stable VPC yielding high titers of up to 5.2 × 10 transducing units/mL (TU/mL). Vectors were concentrated using ultrafiltration devices and upon one freeze-thaw-cycle still routinely yielded titers of > 1 × 10 TU/mL. Formation of replication-competent retroviruses was not detected. However and as a first generation transfer vector was used in this proof-of-concept (POC) study, gag gene sequences were transduced into target cells within a range of 1-10 copies per 1000 genomes indicating the homologous recombination of packaging construct elements with the transfer vector. High yield VPC vector productivity was stable over a couple of months and unintended integration of the transposase gene was not observed. Ecotropic MLV vector particles were demonstrated to efficiently transduce primary murine hematopoietic stem and progenitor cells. This novel concept should foster the future establishment of suspension VPCs.
逆转录病毒载体来源于鼠白血病病毒 (MLV)，是基因治疗方法中最常使用的载体之一，例如造血细胞的基因修饰。目前，载体颗粒主要通过贴壁病毒包装细胞系 (VPC) 生产，这使得生产的扩大化变得繁琐，因此成本高昂。在这里，我们描述了一种快速建立源自嗜性 MLV 的人悬浮 293-F 细胞系的方法。使用转座子载体技术，包装和包膜表达盒以及转移载体有助于建立稳定的 VPC，产生高达 5.2×10 转导单位/毫升 (TU/mL) 的高滴度。使用超滤设备浓缩载体，经过一次冻融循环，仍可常规获得>1×10 TU/mL 的滴度。未检测到复制型缺陷型逆转录病毒的形成。然而，由于在这个概念验证 (POC) 研究中使用了第一代转移载体，gag 基因序列被转导到靶细胞中，每个基因组 1000 个拷贝内的转导拷贝数为 1-10 个，这表明包装构建体元件与转移载体发生了同源重组。高产 VPC 载体生产力在几个月内保持稳定，并且未观察到转座酶基因的非预期整合。嗜性 MLV 载体颗粒被证明能够有效地转导原代小鼠造血干/祖细胞。这个新的概念应该会促进悬浮 VPC 的未来建立。
