The Versatility of 22nt siRNAs and their expanding roles in cancer therapeutics

Gregory Konar

Johns Hopkins University

Publication Date: January 21, 2015

Craig Mello and Andrew Fire’s co-discovery of RNAi in 1998 started much by accident. They thought that injecting an antisense RNA against a specific gene could potentially inhibit mRNA production and thus suppress gene expression. This indeed was true, but something else puzzled them. The injection of their negative control, the sense strand of RNA, also showed a knock down of the gene of interest. Fascinated by this result, they combined the anti-sense and sense RNA strands to form dsRNA (double stranded RNA) complexes and then injected them into Caenorhabditis elegans worms. Their target was the unc-22 gene, a muscle filament protein that, when knocked down, causes the worms to twitch. Indeed, the dsRNA silenced unc-22 gene expression and resulted in the twitching phenotype, leading to the conclusion that the dsRNA was a potent and specific inhibitor of gene expression and that it worked better than either of the single strands on their own (Fire and Mello, 1998).

RNAi is now a very well-known and oft-used method to of inducing site specific gene silencing. dsRNA injected into biological models, either cellular or organismal, interacts with Dicer and/or Drosha complexes present in the cells. Drosha processes only pre-miRNAs and cleaves the lower stem of the hairpin. Dicer interacts with both small interfering (siRNA) and micro-RNA (miRNA) complexes and cleaves the RNAs into pieces 22 nucleotides (nts) in length (Gregory, 2006). These 22 nt pieces then interact with a RNA-induced silencing complex (RISC) which uses argonaute proteins to perform different cellular activities including mRNA cleavage, translation inhibition, and heterochromatin formation (Bagasra, 2004).

Alright, at this point it would probably be assumed that RNA is only useful in silencing gene expression, right? Well, not quite. A mere six years after Mello and Fire’s discovery of the silencing abilities of dsRNA, Long-Cheng Li and his lab showed that dsRNA could also be used for activation of gene expression (Li, 2006). Their discovery, commonly referred to as RNAa, showed that the injection of RNA strands that target the promoter regions of genes can lead to long-lasting effects on the activation of that gene. This activation requires the Ago2 argonaute protein and a histone 3 Lysine-9 methylation (H3-K9 methylation) at the target site for the dsRNA (Li, 2006).

The discoveries of RNAi and RNAa have radically changed the field of genetics. Previously, researchers used scientific methods such as zinc finger nucleases (ZFNs), anti-sense oligonucleotides, and ribozymes for regulation or activation of genes (Phylactou, 1998). These methods, while effective, are not as specific and potent as RNAi or RNAa. The foundation of RNAi and RNAa has led to the development of CRISPR technologies for robust genome editing. CRISPR, or clustered regularly interspaced short palindromic repeats, uses dsRNA injected into a cellular model where it recruits the cas9 protein to create a system that can either sit down in the target site to turn off the gene or, if it targets a promoter region, can activate the target gene (Marraffini, 2011). CRISPR is a revolutionary gene editing technology that helps so many scientists perform experiments with great accuracy and speed and wouldn’t be possible without the advances of Mello, Fire, or Li.

These gene regulation methods are used commonly in determining genes responsible for diseases and cancer. By activating or silencing genes in vitro or in vivo, it becomes possible for researchers to look at whether a gene is silenced in a cancer model, if it aids or abets the proliferation of cancer. If silencing a gene aids in the proliferation of cancer, then the gene can be labelled as a tumor suppressor such as the BRCA1 gene in breast cancer (Duncan, 1998). Likewise, if activating a gene aids in the proliferation of cancer, then the gene is a type of transcription factor. Knowing these tumor suppressors and transcription factors exist allows for further testing that targets these genes. It is important to activate tumor suppressors and silence transcription factors as a means of inhibiting proliferation and growth in cancer. RNA plays a huge role in this research by allowing researchers easy silencing and activation of genes.

Taking RNA research a step further, scientists are looking at the use of siRNAs for targeting genes inside of live mice with intentions of moving towards human models. There exist many therapeutic targets in cancer that are considered to be “undruggable.” Researchers in Dr. Anil Sood’s lab at University of Texas are currently looking into the feasibility of administering siRNAs to specific locations in the body where the siRNAs would then be released and silence the therapeutic target (Wu, 2014). However, there are still many obstacles facing these researchers. Safety is always a prime concern heading into potential clinical testing, and the other major issue is delivery method. Researchers have to take into account the potential degradation of the nanocarrier by nucleases or how the carrier will cross the cell membrane. Getting past these obstacles will unlock the key to drugging the undruggable targets and hopefully lead to more specific cancer treatments.

siRNAs, since their unveiling as a potent biological research agent in the 1990s, have very strong implications in a variety of research roles for the future. These strands of 22nts, sometimes synthesized naturally inside the body and other times created in genomics factories, have uses ranging from detecting new oncogenes and viral causing genes to treating cancer at the source. While they may be short in length, their impact is much larger than their size would suggest. The world of genetic research would be significantly behind without the discovery of these RNAs.

References

1) Bagasra, O., Prilliman, K. (2004). RNA Interference: The Molecular Immune System. Journal of Molecular Histology, 35 (6). 545-553.

2) Duncan, J., Reeves, J., Cooke, T. (1998). BRCA1 and BRCA2 proteins: roles in health and disease. Molecular Pathology, 51 (5). 237-247.

3) Fire, A., Xu, S., Montgomery, M., Kostas, S., Driver, S., Mello, C. (1998). Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 391. 806-811.

4) Gregory, R., Chendrimada, T., Shiekhattar, R. (2006). MicroRNA Biogenesis: Isolation and Characterization of the Microprocessor Complex. Methods in Molecular Biology 342. 33-47.

5) Li, L., Okino, S., Zhao, H., Pookot, D., Place, F., Urakami, S., Enokida, H., Dahiya, R. (2006). Small dsRNAs induce transcriptional activation in human cells. Proceedings of the National Academy of the Sciences, 103 (46). 17337-17342.

6) Marraffini, L., Sontheimer, E. (2011). CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nature Reviews Genetics, 11. 181-190.

7) Phylactou, L., Kilpatrick, M., Wood, M. (1998). Ribozymes as Therapeutic Tools for Genetic Disease. Human Molecular Genetics, 7 (10). 1649-1653.

8) Wu, S., Lopez-Berenstein, G., Calin, G., Sood, A. (2014). RNAi Therapies: Drugging the Undruggable. Science Translational Medicine, 6 (240), 1-7. 

Image taken from blogs.plos.org

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