Howe Geoffrey, Bal Mehtap, Wasmuth Matt, Massaro Giulia, Rahim Ahad A, Ali Sadfer, Rivera Milena, Schofield Desmond M, Omotosho Aminat, Ward John, Keshavarz-Moore Eli, Mason Chris, Nesbeth Darren N
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, London WC1E 6BT, UK.
UCL School of Pharmacy, University College London, London WC1N 1AX, UK.
Mol Ther Methods Clin Dev. 2024 Aug 12;32(3):101317. doi: 10.1016/j.omtm.2024.101317. eCollection 2024 Sep 12.
We sought to engineer mammalian cells to secrete nuclease activity as a step toward removing the need to purchase commercial nucleases as process additions in bioprocessing of AAV5 and AAV9 as gene therapy vectors. Engineering HeLa cells with a serratial nuclease transgene did not bring about nuclease activity in surrounding media whereas engineering serum-free, suspension-adapted HEK293F cells with a staphylococcal nuclease transgene did result in detectable nuclease activity in surrounding media of the resultant stable transfectant cell line, "NuPro-1S." When cultivated in serum-free media, NuPro-1S cells yielded 3.06 × 10 AAV5 viral genomes (vg)/mL via transient transfection, compared with 3.85 × 10 vg/mL from the parental HEK293F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8 × 10 vg/mL from NuPro-1S cells compared with 7.35 × 10 vg/mL from HEK293F cells. AAV9 from both HEK293F and NuPro-1S showed almost identical ability to transduce cells embedded in a scaffold tissue mimic or cells of mouse neonate brain tissue . Comparison of agarose gel data indicated that the DNA content of AAV5 and AAV9 process streams from NuPro-1S cells was reduced by approximately 60% compared with HEK293F cells. A similar reduction in HEK293F cells was only achievable with a 50 U/mL Benzonase treatment.
我们试图对哺乳动物细胞进行工程改造，使其分泌核酸酶活性，以此作为在将腺相关病毒5型（AAV5）和腺相关病毒9型（AAV9）用作基因治疗载体的生物加工过程中，无需购买商业核酸酶作为工艺添加物的第一步。用 Serratia核酸酶转基因对HeLa细胞进行工程改造，并未在周围培养基中产生核酸酶活性，而用葡萄球菌核酸酶转基因对无血清、适应悬浮培养的HEK293F细胞进行工程改造，在所得稳定转染细胞系“NuPro-1S”的周围培养基中确实产生了可检测到的核酸酶活性。在无血清培养基中培养时，NuPro-1S细胞通过瞬时转染产生3.06×10 AAV5病毒基因组（vg）/mL，而亲本HEK293F细胞系为3.85×10 vg/mL。通过超速离心进行纯化后，NuPro-1S细胞产生的AAV9为1.8×10 vg/mL，而HEK293F细胞为7.35×10 vg/mL。来自HEK293F和NuPro-1S的AAV9在转导嵌入支架组织模拟物中的细胞或新生小鼠脑组织细胞方面显示出几乎相同的能力。琼脂糖凝胶数据比较表明，与HEK293F细胞相比，NuPro-1S细胞的AAV5和AAV9工艺流程中的DNA含量降低了约60%。只有用50 U/mL的Benzonase处理，HEK293F细胞才能实现类似的降低。
