Gene Inhibition Therapy
Endogenous RNAi Pathway
The endogenous RNAi pathway can be triggered by different types of double-stranded RNAs.
In this video, we will be reviewing the endogenous or naturally occurring RNA interference (or RNAi) pathway.
The endogenous RNAi pathway can be triggered by different types of double-stranded RNAs. One of these triggers is microRNA18. Endogenous microRNAs play important roles in the biological processes of cell development, differentiation, apoptosis, and proliferation22–25.
A non-coding microRNA gene is usually transcribed by RNA polymerase II to form primary microRNA or “pri-microRNA”18,21. Pri-microRNA is a double-stranded, stem-loop hairpin structure that is cleaved to form precursor microRNA or “pre-microRNA” of approximately 70 to 100 nucleotides, which is then transported into the cytoplasm21,22. Once in the cytoplasm, pre-microRNA loses the stem loop and is further cleaved into shorter duplexes of approximately 22 nucleotides and two 3-prime nucleotide overhangs to form microRNA18,22,26.
MicroRNA then associates with the RNA-induced silencing complex, otherwise known as RISC18,21. Within the microRNA–RISC complex, the microRNA is unwound, releasing the “passenger strand”, while the guide strand—the one that is complementary to the messenger RNA (or mRNA) that is being targeted—remains associated with RISC. This leads RISC to bind to the target mRNA by partial complementarity18,21,27,28.
If the guide strand is only partially complementary to the target mRNA, it can bind to multiple complementary sites or multiple mRNAs, and cause translational repression21,28-30.
Less frequently, when the guide strand is fully or almost fully complementary to the target mRNA, cleavage of the target mRNA occurs21,30,31.
RNAi can also be triggered by small pieces of double-stranded RNA called small interfering RNA, or siRNA21. Although not completely understood, endogenous siRNAs may have roles in repression of transposable elements, chromatin organization, and gene regulation at the transcriptional and post-transcriptional level32,33.
A long double-stranded RNA is first transcribed from cellular genes or infecting pathogens. This RNA is cleaved in the cytoplasm into a smaller double-stranded RNA molecule called siRNA that is 21 to 23 nucleotides long21. siRNA then associates with RISC, and within the siRNA–RISC complex, the siRNA is unwound, releasing and degrading a “passenger strand”. The guide strand, however, remains associated with RISC and leads RISC to bind to the target mRNA18,21,27,28.
To induce gene inhibition, the guide strand must be fully complementary to its target mRNA, which results in gene inhibition via cleavage of the target mRNA21. This summarizes the endogenous RNAi pathway. The next section will discuss key therapeutic triggers of RNAi and how these have been harnessed as therapeutic strategies.
- Wang D, Gao G. Discov Med 2014;18(98):151–161.
- Strachan T, Read AP. Genetic approaches to treating disease: In: Human Molecular Genetics. 5th ed. Florida: CRC Press, 2018:696–699.
- NCBI. Gene silencing. Available at: https://www.ncbi.nlm.nih.gov/probe/docs/applsilencing/. Accessed January 30, 2020.
- Grimm D, Kay MA. Hematology Am Soc Educ Program 2007;2007:473–481.
- Koerner MV, et al. Genes Dev 2018;32(23–24):1514–1524.
- Haussecker D, Kay MA. Science 2015;347(6226):1069–1107.
- Karaki S, et al. Antisense oligonucleotides, a novel developing targeting therapy. In: Antisense Therapy. Available at: https://www.intechopen.com/books/antisense-therapy/antisense-oligonucleotides-a-novel-developing-targeting-therapy. Accessed February 28, 2020.
- Mendonça LS, et al. J Drug Del Sci Tech 2012;22(1):65–73.
- Sliva K, Schnierle BS. Virol J 2010;7:248.
- Wang J, et al. AAPS J 2010;12(4):492–503.
- U.S. FDA. Approved Cellular and Gene Therapy Products. Available at: https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products. Accessed March 27, 2020.
- Wang D, et al. Nat Rev Drug Discov 2019;18(5):358–378.
- Sibley C, et al. Mol Ther 2010;18(3):466–476.
- Wang D, et al. Cell 2020;181(1):136–150.
- Granados-Riveron JT, Aquino-Jarquin G. Cancer Res 2018;78(15):4107–4113.
- Chery J. Postdoc J 2016;4(7):35–50.
- Ozcan G, et al. Adv Drug Deliv Rev 2015;87:108–119.
- Borel F, et al. Mol Ther 2014;22(4):692–701.
- Fire A, et al. Nature 1998;391:806–811.
- The Nobel Prize. Advanced information: RNA interference. Available at: https://www.nobelprize.org/prizes/medicine/2006/advanced-information/. Accessed March 31, 2020.
- Lam JK, et al. Mol Ther Nucleic Acids 2015;4:e252.
- Pushparaj PN, et al. J Dent Res 2008;87(11):992–1003.
- Catalonotto C, et al. Int J Mol Sci 2016;17(10):1712.
- Novák J, et al. Theranostics 2014;4(2):119–133.
- Zampetaki A, et al. Circ Res 2010;107(6):810–817.
- Hydbring P, Badalian-Very G. F1000Res 2013;2:136.
- Chery J. Postdoc J 2016;4(7):35–50.
- Fellmann C, Lowe SW. Nat Cell Biol 2014;16(1):10–18.
- Ramachandran PV, Ignacimuthu S. Appl Biochem Biotechnol 2013;169:1774–1789.
- Herrera-Carrillo E, et al. Hum Gene Ther Methods 2017;28(4):117–190.
- Park JH, Shin C. BMB Rep 2014;47(8):417–423.
- Stein P, et al. PLoS Genet 2015;11(2):e1005013.
- Piatek MJ, Werner A. Biochem Soc Trans 2014;42(4):1174–1179.
- Laganà A, et al. Front Bioeng Biotechnol 2014;2:65.
- Chakraborty C, et al. Mol Ther Nucleic Acids 2017;8:132–143.
- Alnylam Pharmaceuticals. Press release. August 10, 2018. Available at: http://investors.alnylam.com/news-releases/news-release-details/alnylam-announces-first-ever-fda-approval-rnai-therapeutic. Accessed March 31, 2020.
- Onpattro® [package insert]. 2018. Available at: https://www.alnylam.com/wp-content/uploads/pdfs/ONPATTRO-Prescribing-Information.pdf. Accessed March 31, 2020.
- Moore CB, et al. Methods Mol Biol 2010;629:141–158.
- Bofill-De Ros X, Gu S. Methods 2016;103:157–166.
- Myburgh R, et al. Mol Ther Nucl Acids 2014;3:e207.
- Chen X, et al. Cancer Metastasis Rev 2018;37(1):107–124.
- Kurreck J. J RNAi Gene Silencing 2017;13:545–547.
- Rao DD, et al. Adv Drug Del Rev 2009;61:746–759.
- Boudreau RL, et al. Mol Ther 2009;17(1):169–175.
- Fan J, et al. Cancer Gene Ther 2019. doi: 10.1038/s41417-019-0113-y [Epub ahead of print].
- Merlin S, Follenzi AT. Mol Ther Meth Clin Dev 2019;12:223–232.
- Fowler DK, et al. Nucleic Acids Res 2016;44(5):e48.
- Grimm D, et al. Nature 2006;441:537–541.
- Ehlert EM, et al. BMC Neurosci 2010;11:20.
- Giering JC, et al. Mol Ther 2008;16:1630–1636.
- Wu H, et al. PLoS One 2011;6:e28580.
- News Medical Life Sciences. Short Hairpin RNA (shRNA) Interference: Therapeutic Applications. Available at: https://www.news-medical.net/life-sciences/Short-Hairpin-RNA-(shRNA)-Interference-Therapeutic-Applications.aspx#Therapeutic%20applications%20of%20shRNA. Accessed March 31, 2020.
- Wolstein O, et al. Mol Ther Methods Clin Dev 2014;1:11.
- Ahn M, et al. Hum Gene Ther 2011;22(12):1483–1497.
- Ma J, et al. PLoS One 2012;7(10):e46096.
- Stoica L, et al. Ann Neurol 2016;79(4):687–700.
- Wallace LM, et al. Mol Ther 2012;20(7):1417–1423.
- Spronck EA, et al. Mol Ther Methods Clin Dev 2019;13:334–343.
- Wild EJ, Tabrizi S. Lancet Neurol 2017;16(10):837–847.
- Vagner R, et al. Neurol Res Int 2012;2012:358370.