DOI QR코드

DOI QR Code

Validation of Stem-loop RT-qPCR Method on the Pharmacokinetic Analysis of siRNA Therapeutics

Stem-loop RT-qPCR 분석법을 이용한 siRNA 치료제의 생체시료 분석법 검증 및 약물 동태학적 분석

  • 김혜정 (인핸스드바이오) ;
  • 김택민 (인천대학교 생명공학부 나노바이오전공) ;
  • 김홍중 (인핸스드바이오) ;
  • 정헌순 (인핸스드바이오) ;
  • 이승호 (인천대학교 생명공학부 나노바이오전공)
  • Received : 2019.05.07
  • Accepted : 2019.05.28
  • Published : 2019.06.30

Abstract

The first small interfering RNA (siRNA) therapeutics have recently been approved by the Food and Drug Administration in the U.S., and the demand for a new RNA therapeutics bioanalysis method-which is essential for pharmacokinetics, including the absorption, distribution, metabolism, and excretion of siRNA therapeutics-is rapidly increasing. The stem-loop real-time qPCR (RT-qPCR) assay is a useful molecular technique for the identification and quantification of small RNA (e.g., micro RNA and siRNA) and can be applied for the bioanalysis of siRNA therapeutics. When the anti-HPV E6/E7 siRNA therapeutic was used in preclinical trials, the established stem-loop RT-qPCR assay was validated. The limit of detection was sensitive up to 10 fM and the lower limit of quantification up to 100 fM. In fact, the reliability of the established test method was further validated in three intra assays. Here, the correlation coefficient of $R^2$>0.99, the slope of -3.10 ~ -3.40, and the recovery rate within ${\pm}20%$ of the siRNA standard curve confirm its excellent robustness. Finally, the circulation profiles of siRNAs were demonstrated in rat serum, and the pharmacokinetic properties of the anti-HPV E6/E7 siRNA therapeutic were characterized using a stem-loop RT-qPCR assay. Therefore, the stemloop RT-qPCR assay enables accurate, precise, and sensitive siRNA duplex quantification and is suitable for the quantification of small RNA therapeutics using small volumes of biological samples.

SMGHBM_2019_v29n6_653_f0001.png 이미지

Fig. 1. Schematic description of stem-loop RT-qPCR.

SMGHBM_2019_v29n6_653_f0002.png 이미지

Fig. 2. Specific amplification of Anti-HPV E6/E7 siRNA using stem loop RT-qPCR assay.

SMGHBM_2019_v29n6_653_f0003.png 이미지

Fig. 3. Determination of low limit of quantification (LLOQ) concentration.

SMGHBM_2019_v29n6_653_f0004.png 이미지

Fig. 4. Reproducibility and linearity of stem-loop RT-qPCR assay for quantitative detection of siRNA (A-C).

SMGHBM_2019_v29n6_653_f0005.png 이미지

Fig. 5. Quantitative detection of Anti-HPV E6/E7 siRNA in rat serum.

Table 4. Precision of stem-loop RT-qPCR from independent experiments

SMGHBM_2019_v29n6_653_t0001.png 이미지

Table 5. Pharmacokinetic parameter analysis of Anti-HPV E6/ E7 siRNA in rat serum

SMGHBM_2019_v29n6_653_t0002.png 이미지

Table 1. Sequences of siRNA, stem-loop primer and RT-qPCR primers used in the study

SMGHBM_2019_v29n6_653_t0003.png 이미지

Table 2. Stem-loop RT-qPCR results with serial diluted standard siRNA. Comparison of mean Cp value (thrreee replicates) obtained with stem-loop RT-qPCR

SMGHBM_2019_v29n6_653_t0004.png 이미지

Table 3. Robustness of stem-loop RT-qPCR from different researchers

SMGHBM_2019_v29n6_653_t0005.png 이미지

Acknowledgement

Supported by : 중소기업청

References

  1. Burnett, J. C., Rossi, J. J. and Tiemann, K. 2011. Current progress of siRNA/ shRNA therapeutics in clinical trials. Biotechnol. J. 6, 1130-1146.
  2. Cejka, D., Losert, D. and Wacheck, V. 2006. Short interfering RNA (siRNA): tool or therapeutic? Clin. Sci. 110, 47-58.
  3. Chakraborty, C., Sharma, A. R., Sharma, G., Doss, C. G. P. and Lee, S. S. 2017. Therapeutic miRNA and siRNA: Moving from bench to clinic as next generation medicine. Mol. Ther. Nucleic Acids 8, 132-143.
  4. Chen, C. 2005. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33, 79-79.
  5. Fan, A. C., Goldrick, M. M., Ho, J., Liang, Y., Bachireddy, P. and Felsher, D. W. 2008. A quantitative PCR method to detect blood microRNAs associated with tumorigenesis in transgenic mice. Mol. Cancer 7, 74.
  6. Haasnoot, J., Westerhout, E. M. and Berkhout, B. 2007. RNA interference against viruses: Strike and counterstrike. Nat. Biotechnol. 25, 1435-1443. https://doi.org/10.1038/nbt1369
  7. Hannon, G. J. 2002. RNA interference. Nature 418, 244-251.
  8. Hunt, E. A., Broyles, D., Head, T. and Deo, S. K. 2015. MicroRNA detection: Current technology and research strategies. Annu. Rev. Anal. Chem. 8, 217-237.
  9. Jesse, S., Megan, B., Amanda, H., Annaleen, V., Melissa, L. K. and Anja, B. 2017. A new method for generating arrayed RNAi screening tools for any organism. GDI conference. September. London, England.
  10. Jung, H., Rajasekaran, N., Song, S., Kim, Y., Hong, S., Choi, H. and Shin, Y. 2015. Human papillomavirus E6/ E7-specific siRNA potentiates the effect of radiotherapy for cervical cancer in vitro and in vivo. Int. J. Mol. Sci. 16, 12243-12260. https://doi.org/10.3390/ijms160612243
  11. Lam, J. K., Chow, M. Y. T., Zhang, Y. and Leung, S. W. 2015. siRNA versus miRNA as therapeutics for gene silencing. Mol. Ther. Nucleic Acids 4, e252.
  12. Liu, W., Stevenson, M., Seymour, L. W. and Fisher, K. D. 2008. Quantification of siRNA using competitive qPCR. Nucleic Acids Res. 37, e4.
  13. Luo, G. 2002. CYP3A4 induction by drugs: Correlation between a pregnane X receptor reporter gene assay and CYP 3A4 expression in human hepatocytes. Drug Metab. Dispos. 30, 795-804.
  14. Rizk, M. and Tuzmen, S. 2017. Update on the clinical utility of an RNA interference-based treatment: focus on Patisiran. Pharmgenomics Pers. Med. 10, 267-278.
  15. Thakur, A., Fitzpatrick, S., Zaman, A., Kugathasan, K., Muirhead, B., Hortelano, G. and Sheardown, H. 2012. Strategies for ocular siRNA delivery: Potential and limitations of non-viral nanocarriers, J. Biol. Eng. 6, 7.
  16. Tremblay, G. A. and Oldfield, P. R. 2009. Bioanalysis of siRNA and oligonucleotide therapeutics in biological fluids and tissues. Bioanalysis 1, 595-609. https://doi.org/10.4155/bio.09.66
  17. Varkonyi-Gasic, E. and Hellens, R. P. 2011. Quantitative stem-loop RT-PCR for detection of microRNAs. Methods Mol. Biol. 744, 145-157.
  18. Wang, S. J., Wu, S. T., Gokemeijer, J., Fura, A., Krishna, M., Morin, P. and Jemal, M. 2011. Attribution of the discrepancy between ELISA and LC-MS/MS assay results of a PEGylated scaffold protein in post-dose monkey plasma samples due to the presence of anti-drug antibodies. Anal. Bioanal. Chem. 402, 1229-1239.
  19. Zhang, J., Li, X. and Huang, L. 2014. Non-viral nanocarriers for siRNA delivery in breast cancer. J. Control. Release 190, 440-450.