Donohue Nicholas, Li Simeng, Boi Stefano, Rainbow-Fletcher Alana, Barron Niall
National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, A94 X099 Dublin, Ireland.
Pharmaron, 12 Estuary Banks, Speke, Liverpool L24 8RB, UK.
Int J Mol Sci. 2025 Jan 10;26(2):567. doi: 10.3390/ijms26020567.
Recombinant Adeno-associated virus (rAAV) is a popular vector for treating genetic diseases caused by absent or defective genes. rAAVs can be produced that contain a therapeutic transgene, i.e., a correct copy of the affected gene, which is then delivered into target cells. A further application of rAAV is to deliver pro-apoptotic genes such as TNF-related apoptosis-inducing ligand () into cancer cells, leading to tumor regression. However, rAAV production is expensive and insufficient yields may hinder wide-spread adoption especially in systemic conditions. During rAAV production, the therapeutic transgene may be expressed in the producer cell line, and in the case of an oncolytic gene, this would likely lead to cell death thus reducing rAAV yields. Here we demonstrate that expression of TRAIL during rAAV production in HEK293F cells negatively impacts rAAV yield. A shRNA-based strategy was developed to suppress the expression of TRAIL in rAAV-producing cells specifically during the production process. Incorporating a -targeting shRNA expression cassette within the backbone of the rAAV genome-encoding plasmid during triple-transfection of HEK293F cells reduced transgene expression and led to a 60% increase in the yield of rAAV- compared to controls.
重组腺相关病毒（rAAV）是一种用于治疗由缺失或缺陷基因引起的遗传疾病的常用载体。可以生产出包含治疗性转基因（即受影响基因的正确拷贝）的rAAV，然后将其递送至靶细胞。rAAV的另一个应用是将促凋亡基因（如肿瘤坏死因子相关凋亡诱导配体（TRAIL））递送至癌细胞，从而导致肿瘤消退。然而，rAAV的生产成本高昂，产量不足可能会阻碍其广泛应用，尤其是在全身性疾病的治疗中。在rAAV生产过程中，治疗性转基因可能会在生产细胞系中表达，如果是溶瘤基因，这可能会导致细胞死亡，从而降低rAAV产量。在此，我们证明在HEK293F细胞中生产rAAV期间TRAIL的表达会对rAAV产量产生负面影响。我们开发了一种基于短发夹RNA（shRNA）的策略，以在生产过程中特异性地抑制rAAV生产细胞中TRAIL的表达。在HEK293F细胞三重转染期间，将靶向TRAIL的shRNA表达盒整合到rAAV基因组编码质粒的骨架中，可降低转基因表达，并使rAAV-的产量比对照提高60%。
