Ali Sadfer, Rivera Milena, Ward John, Keshavarz-Moore Eli, Mason Chris, Nesbeth Darren N
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, WC1E 7JE, UK.
Heliyon. 2023 Jun 7;9(6):e17067. doi: 10.1016/j.heliyon.2023.e17067. eCollection 2023 Jun.
At present lentiviral vector production for cell and gene therapy commonly involves transient plasmid transfection of mammalian cells cultivated in serum-containing media and addition of exogenous nuclease to reduce host cell and plasmid DNA impurities. Switching from serum-containing media to chemically-defined, serum free media, and minimising the number of process additions, are both increasingly regarded as necessary steps for simplifying and potentially automating lentiviral vector bioprocessing in future. Here we adapted human embryonic kidney 293T (HEK293T) cells to grow in serum-free media and also modified these cells with transgenes designed to encode a secreted nuclease activity. Stable transfection of HEK293T cells with transgenes encoding the nuclease B (NucB) open reading frame with either its native secretion signal peptide, the murine Igκ chain leader sequence or a novel viral transport fusion protein, all resulted in qualitatively detectable nuclease activity in serum-free media. Serum-free transient transfection of human embryonic kidney HEK293T cells stably harbouring the transgene for NucB with its native secretion signal produced active lentivirus in the presence of medium-resident nuclease activity. This lentivirus material was able to transduce the AGF-T immortal T cell line with a green fluorescent protein reporter payload at a level of 2.05 × 10 TU/mL (±3.34 × 10 TU/mL). Sufficient nuclease activity was present in 10 μL of this unconcentrated lentivirus material to degrade 1.5 μg DNA within 2 h at 37 °C, without agitation - conditions compatible with lentivirus production. These observations demonstrate that lentiviral vector production, by transient transfection, is compatible with host cells harbouring a nuclease transgene and evidencing nuclease activity in their surrounding growth media. This work provides a solid basis for future investigations, beyond the scope of this present study, in which commercial and academic groups can apply this approach to therapeutic payloads and potentially omit exogenous nuclease bioprocess additions.
目前，用于细胞和基因治疗的慢病毒载体生产通常涉及在含血清培养基中培养的哺乳动物细胞的瞬时质粒转染，并添加外源性核酸酶以减少宿主细胞和质粒DNA杂质。从含血清培养基切换到化学成分明确的无血清培养基，并尽量减少工艺添加物的数量，越来越被视为未来简化和可能自动化慢病毒载体生物加工的必要步骤。在这里，我们使人类胚胎肾293T（HEK293T）细胞适应在无血清培养基中生长，并还用设计用于编码分泌型核酸酶活性的转基因对这些细胞进行了修饰。用编码核酸酶B（NucB）开放阅读框的转基因对HEK293T细胞进行稳定转染，该转基因带有其天然分泌信号肽、鼠Igκ链前导序列或一种新型病毒转运融合蛋白，所有这些都导致在无血清培养基中可定性检测到核酸酶活性。用带有天然分泌信号的NucB转基因稳定转染的人类胚胎肾HEK293T细胞在无血清条件下进行瞬时转染，在存在培养基中固有核酸酶活性的情况下产生了活性慢病毒。这种慢病毒材料能够以2.05×10 TU/mL（±3.34×10 TU/mL）的水平用绿色荧光蛋白报告基因载荷转导AGF-T永生T细胞系。在37℃下，10μL这种未浓缩的慢病毒材料中存在足够的核酸酶活性，在2小时内无需搅拌即可降解1.5μg DNA - 这些条件与慢病毒生产兼容。这些观察结果表明，通过瞬时转染生产慢病毒载体与携带核酸酶转基因并在其周围生长培养基中显示核酸酶活性的宿主细胞兼容。这项工作为未来超出本研究范围的研究提供了坚实的基础，商业和学术团体可以将这种方法应用于治疗性载荷，并有可能省略外源性核酸酶生物加工添加物。
