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59 Preclinical platform for nucleic acid drug evaluation: siRNA and mRNA therapeutics
  1. Dong Wang1,
  2. Xiangnan Qiang2,
  3. Zhixiang Zhang1 and
  4. Qingyang Gu3
  1. 1WuXi AppTec, Shanghai, China
  2. 2WuXi AppTec, Cambridge, MA, USA
  3. 3WuXi AppTec, Shanghai, Jiangsu, China
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Since the emerging of COVID-19 pandemic, numerous nucleic-acid therapeutics have been approved for clinical use. Distinguishing themselves from conventional protein-targeted therapy, nucleic acid therapeutics offer the potential for sustained effects via gene modification or inhibition. This class of therapeutics can be further categorized divided into oligonucleotide, single-guide RNAs (sgRNA), and messenger RNAs (mRNA). In this study, we evaluated the anti-tumor activity of PKN3-targeted siRNA and mRNA-encoded CD3-EpCAM BsAb.

Methods To identify suitable siRNA candidates, we utilized psiCHECK system for screening, and performed qPCR and western blot assays to confirm the inhibition of target expression and loss of function, both in vitro and in vivo. The potential off-target effect of siRNA was determined through RNA sequencing. The in vivo efficacy of these siRNA was assessed by using subcutaneous tumor xenograft models. Serum biochemistry analysis and histological examination, such as HE staining, of various organs were conducted for the safety evaluation of siRNA. Moreover, the concentration of siRNA in blood of tumor-bearing mice was measured and quantified by LC-MS.

Regarding the mRNA-encoded test article, we conducted flow cytometry analysis to determine the target expression levels. The dynamic concentration of test article in tumor cells after transfection was measured by qPCR and flow cytometry. To evaluate the anti-tumor activity and immune activation effect of mRNA-encoded BsAb in vitro, flow cytometry and ELISA were further employed. We also performed IVIS imaging to assess the bio-distribution of mRNA-encoded BsAb in vivo. And the efficacy was evaluated by using subcutaneous tumor xenografts, in which the mRNA content in tumor-bearing mice was examined by qPCR and the early safety of mRNA-encoded BsAb was determined through serum biochemistry analysis and HE staining of carious organs.

Results PKN3-targeted siRNA demonstrated significant inhibition of tumor growth without exhibiting any discernible off-target effects. Serum biochemistry and tissue staining analysis further supported the safety profile of siRNA in vivo. Furthermore, mRNA-encoded BsAb promoted the activation and cytotoxicity of human T cells, and exhibited pronounced inhibition effect of tumor growth in vivo while maintaining a favorable safety.

Conclusions In this study, we conducted a comprehensive evaluation of the activity and anti-tumor effects of PKN3-targeted siRNA and mRNA-encoded CD3-EpCAM BsAb. Our findings provide compelling evidence suggesting that the therapeutic strategies utilizing nucleic acid hold significant research value and exhibit promising potential for clinical applications.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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