For a disease to be thoroughly defeated, its early diagnosis should be coupled with the pertinent treatment, which opens to yet another world of possibilities available where miRNAs can once again take the spotlight. This is because, as mentioned previously, these tiny ncRNAs have been shown to be involved in the regulation of mRNA expression as well as they’re able to reprogram molecular pathways in diseases like cancer (Çiğir & Yusuf, 2014) and such feat doesn’t limit itself to be exploited for disease diagnosis, but also in the field of disease therapy given that they can be therapeutic targets that may either influence drug resistance for a disease or become drugs themselves (Çiğir & Yusuf, 2014).
miRNA therapeutics offer optimism for the future of medicine, hence it’s a field also worth diving deep into, given that a therapy based on the delivery of RNA-carrying vesicles might just offer a simpler and safer alternative to, for instance, stem-cell therapy; such advantage extends itself to the treatment of even neurological disorders as long as researchers find ways to deliver such RNA-carrying vesicles across the blood-brain barrier (Herb, 2020).
Also, current therapeutic targets are mostly proteins that belong to either one of the following three protein classes: enzymes, receptors and transporters. This has put a burdensome challenge to research and drug discovery given the large amount of resources needed to be invested (money in the scale of billions of dollars and longitudinal studies that may span decades), coupled with the natural challenges that drugs’ R&D carry with themselves (the need to follow Lipinski’s Rule of Five[1] as well as making the drug actually modulate the target protein) and the very strict regulatory mechanisms that ensure drug safety; all in all, the end result is that the number of protein drug targets is limited; although the human genome encodes 100,000–200,000 proteins, it has been shown that only 207 proteins are targeted by FDA-approved small-molecule drugs. Furthermore, it is estimated that only 600 disease-modifying protein drug targets exist (Schmidt, November, 2014). Such barriers then prompt researchers to shift focus to alternative molecules, such as circulating RNAs, especially miRNAs, due to their useful qualities already mentioned, such as availability in exosomes in the bloodstream that makes its research easy, therefore facilitating the search for drug targets, or its critical role in gene silencing or expression, therefore allows a greater quantity of possible proteins to be able to be indirectly modulated.
Another advantage is that given how miRNA biomarkers can be employed to develop a particular miRNA expression profile for a particular disease, this can also open the possibility of a personalized therapy based on such miRNA signature through either delivery of antisense miRNA strands to inhibit specific upregulated miRNAs or miRNA mimics to balance out specific downregulated miRNAs; Table 1 summarizes some companies that are currently developing such strategies to diseases like cancer, HCV infection or heart failure. miRNA therapeutics hence may contribute to the foundations of personalized medicine by allowing targeted therapies, which are needed in disease like cancer so that specific oncogenic pathways are interfered, thus improving patient survival and care.
Finally, miRNA deregulation may not only affect pathogenesis, but also influence in drug resistance. A number of published reports show that manipulating expression of specific miRNAs can alter the drug sensitivity or that miRNAs are themselves biologically involved in the body’s resistance response, and this kind of phenomenon doesn’t restrict itself to cancer pathologies, but is also associated with drug resistance in treatment of conditions such as epilepsy, multidrug-resistant tuberculosis, and insulin sensitivity (Hanna, Hossain, & Kocerha, 2019).
Given such premise that shows yet another potential of miRNAs, now in the field of therapeutics, it is worth exploring how miRNAs can be delivered to the patient. There are several strategies that target putative miRNAs to treat a disease. Among them, there are the following:
Delivery of miRNAs mimics or antisense oligonucleotides
A therapy based on the delivery of nucleotides is an active area of drug development designed to treat a variety of gene-specific diseases (Xuan, Philip, & Thomas, 2017 ). Oligonucleotides can either take the role of miRNA mimics to aid the host’s miRNAs in regulating gene expression or be designed as anti-sense oligonucleotides (ASO) to inhibit miRNAs (Schmidt, November, 2014). To view what are some of the current progresses made towards this area, please examine (Schmid, 2017) or (Schmidt, November, 2014) and Table 1:
Table 1: Summary of the progresses made towards the introduction of miRNA antisense inhibitors or miRNA mimics designed to treat diverse diseases
Source: (Drug target miRNAs: chances and challenges, November, 2014) by Marco F. Schmidt
The use of oligonucleotides is inspired by the fact that while a deregulation of miRNAs has been causally correlated with a disease’s development, a compensation for such deregulation (for instance, inhibiting upregulated miRNA or enhancing downregulated miRNA) has also been shown, mostly in animal models or cellular assays, to alleviate the phenotype expressed by certain disease. For instance, in an infectious disease like tuberculosis, Lou et al. (2017) demonstrated that the injection of miR-20b mimics to compensate for its downregulation in TB can alleviate the inflammatory response in TB mice (Lou, Wang, Zhang, & Qiu, 2017). Another example is upregulated miR – 802 in obese mouse models, which has been shown to impair insulin transcription and secretion, which in humans is related to the development of type 2 diabetes. Zhang et al. studied how inhibition of miR – 802 might influence pancreatic β cells, so they knocked down miR-802 in primary islets and Min6 cells and they interestingly found out a significant increment in the expression of insulin at mRNA, protein levels and insulin content (Fangfang, et al., 2020). These are just a few examples of how researchers have found out how the compensation for deregulated miRNAs can alleviate or stop pathogenesis of a disease, for more, please visit (Ramiro, Guido, & Carlo, 2014), where experimental data concerning miRNAs and how they influence cancer progression is shared. Table 2 contains such data.
Table 2: Experimental data showcasing how the compensation of deregulated miRNAs can alleviate disease progression of cancer.
Of course, it’s worth mentioning that these research results have been limited into either experimenting with animal models or cell lines, so they don’t necessarily translate into human models; nonetheless, an overall idea is that there are clear prospects to explore furthermore in the field miRNA therapeutics and strategies to compensate for a miRNA deregulation to alleviate the symptoms of a disease.
Also, the delivery of oligonucleotides, such as with liposomal nanoparticles, is a field that requires further research and development in order to overcome pharmacokinetic challenges and issues like poor permeability to a cell’s insides, where the target miRNA is located (Schmid, 2017). There are also other more down-to-earth questions that need to be raised, such as if the actual miRNA – targeting drug is developed, then for how long can it compensate the deregulated miRNA before it becomes deregulated again or at what prize would it be available for the general populace. Because the drugs shared previously are still in clinical trials, it is still too early and difficult to give a definite answer to the above concerns other than to provide some insight about a possible response. For example, in 2008 Horwich and Zamore published a protocol for the design and delivery of antisense oligonucleotides to block microRNA function in cultured Drosophila and human cells; they report that their ASOs could provide at least 7 days of miRNA inhibition from a single transfection (Horwich & Zamore, 2008).
Disruption or enhancement of a miRNA’s pathway of biogenesis
There are several strategies designed to modulate target miRNA’s concentrations, and before going through these strategies it’s worth first getting an idea of the causes of miRNA dysregulation (please examine (Marilena & Carlo, 2012)) as that’d provide insight into why certain molecules are chosen as therapeutic targets; in short, miRNA therapeutics can be focused on intervening in its biogenesis, on the field of epigenetics or targeting the corresponding DNA loci.
For instance, in miRNA biogenesis, as shown in Figure 1, molecules can intervene during DICER-mediated pre-miRNA processing in order to either upregulate a miRNA through enhancing its maturation, otherwise inhibit its maturation, thus reducing its expression levels. Targeting pre-miRNA comes with its advantage due to its relative bigger structure than miRNA, which increases its ligandability, thus increasing the change of finding a molecule to attach to them (Schmidt, November, 2014).
Figure 1: An illustration of how small bioactive molecules can either enhance or inhibit a target miRNA during the DICER-mediated processing step.
Source: (Drug target miRNAs: chances and challenges, November, 2014)
An alternative to the above is targeting the miRNA-AGO2 complex through anti-miR-AGOs. In theory, these should have better pharmacokinetic properties than oligonucleotides given that they don’t target miRNAs per se, but rather AGO2 protein’s active site with the miRNA of interest identified through a specific oligonucleotide sequence, which altogether leads to the development of more hydrophobic molecules with lower molecular weight than the mentioned oligonucleotide inhibitors (Schmidt, November, 2014).
Epigenetics also offer an alternative for miRNA therapeutics. This is considering the reported evidence of how microRNA expression can be affected by changes in the epigenetic program. In theory, the existence of epigenetic drugs, such as DNA demethylating agents and histone deacetylase inhibitors, which are able to reverse an aberrant methylation or acetylation status, raises indeed the intriguing possibility to regulate microRNA levels, for example to restore the expression of tumor suppressor microRNAs, thus reverting a tumoral phenotype (Marilena & Carlo, 2012).
miRNA editing: miRNA and CRISPR
DNA may also be a therapeutic target regulating miRNAs. Given the high prospects displayed by the CRISPR – CAS13a system in nucleic acid detection, the CRISPR-CAS system wouldn’t be exempt from also being a therapeutic platform with great potential to be harnessed, given that it may compensate for the flaws of an oligonucleotides-based approach.
For example, Chang et al. employed the CRISPR-cas9 (Figure 2)[2] system with single-guide RNAs specifically targeting DNA loci expressing the biogenesis processing sites of selected microRNAs (i.e. miR-17, miR-200c, and miR-141) to see if this can repress such miRNA’s expression. Among the benefits seen employing the CRISPR/cas9 system over the oligonucleotides approach are higher specificity and robustness. For instance, the team reports that DNA sequencing results showed that CRISPR/cas9 editing could unbiasedly occur if less than 2 mismatches were identified between the target sgRNA; of course, 2 mismatches are still significant as noted by the researchers given that off-target effects on miRNA regulation may result in unwanted gene expression or silencing, which may prove harmful for the host. Nonetheless, from this, an advantage that can be extrapolated is that CRISPR/Cas9 seems more capable of knocking down miRNA through its corresponding DNA loci given that, as said before, miRNAs themselves are very short in length, which has resulted in challenges for ASOs to actually bind to them (ligandability). Another advantage is that the team reported that, for instance, miR-17 was continuously downregulated in the cells with CRISPR/cas9 targeting miR-17 over the course up to 30 days after transfection (Chang, et al., 2016).
Of course, one such limitation that becomes apparent is that CRISPR-CAS9 seems to be focused only on knocking down a gene loci that leads to the downregulation of a desired miRNA, however, there needs to be further development in the area of how a CRISPR-Cas system can also upregulate miRNAs, as research has shown that upregulation of selected miRNAs can also reverse the phenotype of a disease, as shared previously (please revisit Table 1).
Figure 2: CRISPR-CAS9 System targeting a specific DNA loci that transcribes a particular miRNA
Source: (Chang, et al., 2016)
All in all, it could be the case that with enough research and development, the CRISPR/Cas9 system, or maybe another system that employs a novel nuclease yet to be discovered, could be a more efficient, specific, economic, convenient, and stable technology for knocking down miRNA, so that such therapeutic platform can be accessible for the general population instead of very technical settings like laboratories, limiting miRNA therapeutics to researchers only.
Dietary miRNA
“Let food be thy medicine and medicine be thy food” said Hippocrates. A disease prognosis is done better if coupled with a lifestyle that takes preventive measures towards a disease. Because it’s more than well shown that miRNAs regulate gene expression, their presence in exosomes in food like milk, ginger, mushrooms, grapes, etc., have attracted researchers’ attention to see how such RNA-carrying extracellular vesicles can affect the host, particularly the gut microbiome[3]. If research in this field is ripened, and by ripened we mean a thorough understanding of the gut microbiome and how miRNA carrying-exosomes interact with each other, theoretically, it’s possible to bioengineer exosome-like nanoparticles derived from plants or the food mentioned above for them to carry RNA (and of course maybe proteins and lipids) for therapeutic purposes for certain disease (Campbell, 2020). A particular example of a theoretical scenario where a particular miRNA is delivered to a host, for instance, would be the addition of this molecule to infant milk formula to improve the baby’s health; such view is shared by Steven Hicks, a pediatrician at Pennsylvania State University College of Medicine in Hershey, who led a team in a trial which started in 2018 that measured levels of miRNA found in breast milk, infant saliva and infant stool over 12 months, also tracking the health of infants — specifically whether they develop food allergies, eczema or asthma. He said that his group is looking for associations between high levels of particular strands of miRNA in breast milk that survive digestion and protective effects against such conditions in babies (Nguyen, 2020).
Challenges of miRNA therapeutics
Just as how challenges arose from miRNA diagnostics, miRNA therapeutics also shows a lot of barriers to surpass. Despite the many opportunities that may burst forth from the ongoing research about ncRNAs, particularly miRNAs, there are diverse challenges, some of which have been shared sporadically in the sections above, such as the design of ASOs high ligandability and vesicles of high permeability.
Also, perhaps an advantageous, yet also an inconvenient feature of miRNAs is their ability to simultaneously target multiple genes, which has made them an attractive alternative to the ‘one target, one drug’ model, but it also decreases their overall disease specificity and increases the likelihood of potential off target effects (Marissa & Scott, 2020).
Another such double-edged-sword quality is miRNA’s great tissue/cell specificity, which raises a challenge for drug makers because the therapy must be targeted not only towards a specific miRNA, but a specific miRNA of a particular cell type, as noted by researcher Andrew Baker, a molecular biologist at the University of Edinburgh. This was regarding a miRNA he researched, miR-21, which was associated with plaque build-up in arteries and have been found seven times more abundant in peripheral arteries clogged with atherosclerosis than in healthy arteries. He noted that miR-21 is expressed in virtually every cell. If there is a broad, unfocused attempt to suppress such omnipresent molecule, then once again off target effects with unintended health consequences might occur (Woolston, 2019).
Also, as with miRNA being as biomarkers, it is always worth noting miRNA therapeutics is not complete if miRNA is the only point of focus. Once again, phenotype is the result of genotype and environmental factors, so for a disease therapy to be thorough, changes in the environment, lifestyle, diet, among other factors, must be taken into account.
As for drug targets, it’s also worth saying that miRNAs shouldn’t be treated as the only molecule to invest the future on. Protein, lipids, other circulating, noncoding-RNAs or molecules yet to be discovered all can become better therapeutic targets or biomarkers to treat diseases that nowadays ravages humanity, whether it is a cancer or pathogen-driven.
However, all in all, miRNAs seem to be a possible candidate to becoming the next blockbuster in medicine, with high prospects of revolutionizing (maybe together with the CRISPR-CAS system) disease prognosis and therapy.
Afterthoughts
Despite sharing some findings regarding miRNAs and their role in disease prognosis and therapy, may main goal is to inspire the reader to choose a path of R&D for his or her future. I also want to share a view regarding how artificial intelligence can play a pivotal role for bringing such technology to each household in order to democratize medicine and allow the general population to become healthier.
Such view is based on a product small and easy to use so that the average household can have access to it and employ it for a constant monitoring of the family members’ miRNA expression patterns.
1. It may resemble an apparatus similar to miRoculus (or maybe miROculus themselves) or maybe MinION (Figure 3)[4] designed by Oxford Nanopore Technologies, which is a very small device used to perform DNA or RNA sequencing.
Figure 3: Screenshot of the MinION
2. Whether the device is like miROculus or MinION, the inner architecture can be adapted from either the CRISPR-LbuCas13a detection platform by (Yuanyue, Xiaoming, Ru, & Da, 2019) or the CRISPR/Cas13a-Powered electrochemical microfluidic biosensor by (Richard, et al., 2019), so that a miRNA diagnostics can be performed from a simple blood sample loaded to this small device. This is in order to reduce the cost of reagents as wells as facilitate the procedure of diagnosis.
3. The device is then connected to a terminal, maybe a PC or personal laptop, to upload data about the identified miRNA expression profile. This data could also be shared through the cloud with a medical team or professional for a more thorough analysis. Either way, such data, coming from a blood sample that underwent CRISPR-CAS diagnostics for identifying a miRNA expression pattern, will act as input towards a deep neural network that’s able to prognose a disease from such. For a depiction of a possible network that performs such miRNA-disease associations, please examine (Marissa & Scott, 2020), where they share an implementation of a MiRNA-disease Association Prediction (MAP) network that’s constructed by combining miRNA-gene associations, protein-protein interactions (PPI), and gene-disease associations collected from databases to form a heterogeneous tripartite network that’s then reduced to a miRNA-disease bipartite network through a network diffusion algorithm that performs a random walk starting at the known disease genes to rank miRNA-disease associations (Figure 4). The output, hence, can resemble Figure 5, given that’ll be user friendly and easier to understand.
Figure 4: Summary of the architecture of the MAP network
Source: (Predicting miRnA-based disease-disease relationships through network diffusion on multi-omics biological data, 2020) by Marissa & Scott
Figure 5: Output of the system described above for an early diagnostics of disease.
I envision that an addition to such network would be the prediction of also putative miRNA targets that may aid the relevant medical personnel to design the most appropriate therapy (which may also be based on a CRISPR-Cas system). Also, the network can be further trained to prognose a disease from other covariates such as age, gender, ethnicity, gut micriobiome composition, among others to perform a more precise diagnosis.
All in all, as said before, miRNAs can be next blockbuster in medicine that may aid in revolutionizing the field of diagnostics and therapy.
Bibliography
Campbell, K. (2020, June 17). nature Outlook. Retrieved August 24, 2020, from Do the microRNAs we eat affect gene expression?: https://www.nature.com/articles/d41586-020-01767-x
Chang, H., Bin, Y., Ruixia, M., Xiaoguo, Z., Hongyou, Z., & Yaguang, X. (2016). CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo. SCIENTIFIC Reports, 6:22312.
Christopher, A. L., Franco, L., Beryl W., D., & Paul J., F. ((2001) ). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews 46 , 3–26.
Çiğir, B. A., & Yusuf, B. (2014). Chapter 19: Use of MicroRNAs in Personalized. In Y. Editors: Malik, & A. Jens, miRNomics: MicroRNA Biology and Computational Analysis. Springer New York Heidelberg Dordrecht London.
Fangfang, Z., Dongshen, M., Wanli, Z., Danwei, W., Tingsheng, L., Yuhong, L., . . . Liang, J. (2020). Obesity-induced overexpression of miR-802 impairs insulin transcription and secretion. nature COMMUNICATIONS, 11:1822.
Hanna, J., Hossain, G., & Kocerha, J. (2019). The Potential for microRNA Therapeutics and Clinical Research. frontiers in Genetics, 10:478.
Herb, B. (2020, June 17). nature OUTLOOK. Retrieved August 22, 2020, from Extracellular RNA: https://www.nature.com/articles/d41586-020-01762-2
Horwich, M. D., & Zamore, P. D. (2008). Design and delivery of antisense oligonucleotidesto block microRNA function in cultured Drosophilaand human cells. NATURE PROTOCOLS, VOL.3 NO.10 1536 - 1549.
Lou, J., Wang, Y., Zhang, Z., & Qiu, W. (2017). MiR-20b inhibits mycobacterium tuberculosis induced inflammation in the lung of mice through targeting NLRP3 . Experimental cell research, 358(2):120-128.
Marilena, V. I., & Carlo, M. C. (2012). Causes and Consequences of microRNA Dysregulation. Cancer J , 18(3): 215–222.
Marissa, S., & Scott, t. W. (2020). Predicting miRnA-based disease-disease relationships through network diffusion on multi-omics biological data. Scientific Reports | nature research, 10:8705.
Nguyen, T. (2020, June 17). nature OUTLOOK. Retrieved August 24, 2020, from Unravelling the mysteries of microRNA in breast milk: https://www.nature.com/articles/d41586-020-01768-w
Ramiro, G., Guido, M., & Carlo, M. C. (2014). Targeting MicroRNAs in Cancer: Rationale, Strategies and Challenges. Nature Reviews Drug Discovery, 9(10): 775–789.
Richard, B., Julia, B., Claire, C., Mailin, M., Sibylle, M., Wilfried, W., . . . Gerald, A. U. (2019). CRISPR/Cas13a-Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification-Free miRNA Diagnostics. Advanced Materials, 31, 1905311.
Schmid, M. F. (2017). Drug Target miRNA | Methods and Protocols. New York : Springer Science+Business Media .
Schmidt, M. F. (November, 2014). Drug target miRNAs: chances and challenges. Review Cell Press, Trends in Biotechnology,, 578-586.
Sharda, T., & Kalpana, G. (2015). miR-122 is a Unique Molecule with Great Potential in Diagnosis, Prognosis of Liver Disease, and Therapy Both as miRNA Mimic and Antimir. Curr Gene Ther. , 15(2): 142–150. .
Synthego. (2019, 08 29). Resources. Retrieved 08 24, 2020, from CRISPR 101 (ebook): https://www.synthego.com/resources/crispr-101-ebook
Woolston, C. (2019, October 16). nature OUTLOOK. Retrieved August 14, 2020, from How RNA therapies could be used to tackle the world's bigest kller: https://www.nature.com/articles/d41586-019-03073-7
Xuan, C., Philip, G., & Thomas, P. (2017 ). Safety of antisense oligonucleotide and siRNA-based therapeutics. Drug Discov Today, 22(5):823-833.
Yuanyue, S., Xiaoming, Z., Ru, H., & Da, X. (2019). High-Fidelity and Rapid Quantification of miRNA Combining crRNAProgrammability and CRISPR/Cas13atrans-Cleavage Activity. Analytical Chemistry, 91, 5278−5285.
[1] A set of rules that’d enable a drug to be assimilated by the body in order to target a protein. Such drug should have the following qualities: no more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight is no greater than 500 and the calculatedLog P (CLogP) is no greater than 5 (or MlogP.4.15) (Christopher, Franco, Beryl W., & Paul J., (2001) ). [2] Cas9 differs from Cas13. The former nuclease is guided by an sgRNA towards a target sequence in the DNA; subsequently, Cas9 makes a double strand break in the DNA that’s followed by endogenous repair mechanisms that may result in gene knockout via frameshift mutation or gene knock in if a DNA template is present (Synthego, 2019). Though the phase of recognition of a target sequence is similar, the latter nuclease’s cleavage mechanism is exploited to indicate the presence of a desired nucleic acid. [3] There has been recent attention focused on the gut microbiome; for a deeper insight please visit: https://www.nature.com/collections/eccfeecfae [4] Please visit: https://nanoporetech.com/products/minion
Comments