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 × 10⁶ transducing units/mL (TU/mL). Vectors were concentrated using ultrafiltration devices and upon one freeze-thaw-cycle still routinely yielded titers of > 1 × 10⁶ 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.

源自鼠白血病病毒 (MLV) 的逆转录病毒载体是基因治疗方法（例如造血细胞的基因修饰）中最常用的载体之一。目前，载体颗粒主要是使用贴壁病毒包装细胞系 (VPC) 生产的，这使得生产规模扩大很费力，因此成本很高。在这里，我们描述了人悬浮 293-F 细胞系衍生的亲嗜性 MLV VPC 的快速建立。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 ^{6的高滴度}转导单位/mL (TU/mL)。使用超滤装置浓缩载体，并且在一次冻融循环后仍常规产生 > 1 × 10 ⁶ TU/mL 的滴度。没有检测到具有复制能力的逆转录病毒的形成。然而，作为概念验证 (POC) 研究中使用的第一代转移载体，gag基因序列被转导到靶细胞中，范围为每 1000 个基因组 1-10 个拷贝，表明包装构建元件与转移载体的同源重组。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意外整合。亲嗜性 MLV 载体颗粒被证明可以有效地转导原代小鼠造血干细胞和祖细胞。这个新颖的概念应该促进未来建立暂停 VPC。