中国仓鼠卵巢 (CHO) 细胞被广泛认为是工业重组蛋白制造的首选宿主系统，在开发药物（包括抗癌疗法）中发挥着至关重要的作用。然而，基于哺乳动物细胞的生物制药生产方法仍然受到细胞限制的困扰，例如生长有限和生产力低下。 MicroRNA-21 (miR-21) 对多种恶性肿瘤有重大影响，包括多形性胶质母细胞瘤 (GBM)。然而，生产力和生长速度的降低与 CHO 细胞中 miR-21 的过度表达有关。当前的研究旨在利用 CRISPR 介导的精确整合到靶染色体 (CRIS-PICh) 系统以及 Bxb1 重组酶介导的盒式交换 (RMCE) 来工程化重组 CHO (rCHO) 细胞，以表达环状 miR-21 诱饵。 CM21D）具有五个凸起的 miR-21 海绵结合位点。实施核糖核蛋白 (RNP) 递送方法，利用 CRIS-PITCH 技术将着陆垫插入基因组中。随后，使用 RMCE/Bxb1 系统将两侧为 Bxb1 attB 的 CM21D 暗盒重新定位到集成着陆垫中。该策略将靶向效率提高了1.7倍，并减少了脱靶效应。 miR-21 靶基因（Pdcd4 和 Atp11b）注意到 miR-21 通过 CM21D 海绵化后表达显着增加。表达 CM21D 后，rCHO 细胞的倍增时间显着缩短，生长速度增加 1.3 倍。进一步分析显示，分泌型重组蛋白 hrsACE2 的产量增加了 2.06 倍。因此，我们可以得出结论，海绵诱导的 miR-21 抑制可能导致生长速率增加，这可能与 CHO 细胞生产力的增加有关。对于包括 CHO 细胞在内的工业细胞系，生产力的提高至关重要。我们的研究结果表明CM21D是一种吉祥的CHO工程方法。要点： • CHO 是生产工业治疗药物的理想宿主细胞系，miR-21 在产生重组蛋白的 CHO 细胞中下调。 • miR-21 靶基因注意到，miR-21 通过CM21D 海绵化后表达显着增加。此外，CM21D 对 miR-21 的海绵作用增强了 CHO 细胞的生长速度。 • CM21D 敲入后，表达重组 hrs-ACE2 蛋白的 CHO 细胞的生产力和生长率有所提高。© 2024。作者。

Chinese hamster ovary (CHO) cells, widely acknowledged as the preferred host system for industrial recombinant protein manufacturing, play a crucial role in developing pharmaceuticals, including anticancer therapeutics. Nevertheless, mammalian cell-based biopharmaceutical production methods are still beset by cellular constraints such as limited growth and poor productivity. MicroRNA-21 (miR-21) has a major impact on a variety of malignancies, including glioblastoma multiforme (GBM). However, reduced productivity and growth rate have been linked to miR-21 overexpression in CHO cells. The current study aimed to engineer a recombinant CHO (rCHO) cell using the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system coupled with the Bxb1 recombinase-mediated cassette exchange (RMCE) to express a circular miR-21 decoy (CM21D) with five bulged binding sites for miR-21 sponging. Implementing the ribonucleoprotein (RNP) delivery method, a landing pad was inserted into the genome utilizing the CRIS-PITCh technique. Subsequently, the CM21D cassette flanked by Bxb1 attB was then retargeted into the integrated landing pad using the RMCE/Bxb1 system. This strategy raised the targeting efficiency by 1.7-fold, and off-target effects were decreased. The miR-21 target genes (Pdcd4 and Atp11b) noticed a significant increase in expression upon the miR-21 sponging through CM21D. Following the expression of CM21D, rCHO cells showed a substantial decrease in doubling time and a 1.3-fold increase in growth rate. Further analysis showed an increased yield of hrsACE2, a secretory recombinant protein, by 2.06-fold. Hence, we can conclude that sponging-induced inhibition of miR-21 may lead to a growth rate increase that could be linked to increased CHO cell productivity. For industrial cell lines, including CHO cells, an increase in productivity is crucial. The results of our research indicate that CM21D is an auspicious CHO engineering approach. KEY POINTS: • CHO is an ideal host cell line for producing industrial therapeutics manufacturing, and miR-21 is downregulated in CHO cells, which produce recombinant proteins. • The miR-21 target genes noticed a significant increase in expression upon the miR-21 sponging through CM21D. Additionally, sponging of miR-21 by CM21D enhanced the growth rate of CHO cells. • Productivity and growth rate were increased in CHO cells expressing recombinant hrs-ACE2 protein after CM21D knocking in.© 2024. The Author(s).