What are microRNA's, and How Did We Get Here? The discovery of microRNA is an interesting story intimately related to C. Elegans, an animal model used for investigation of animal development. C. Elegans is a very small nematode which lives in garden soil, feeding on bacteria. The number of papers published utilizing this creature as an animal model for human biology has increased substantially after 1990. They are easy to grow, small, transparent and have a short life cycle. Individuals within the species are invariant, with less than 1000 total cells. Drs. Victor Ambros and Gary Ruvkun, with Harvard University and Massachusetts General Hospital, originally discovered microRNA’s using C. Elegans while studying abnormalities in early development. Dr. Ambros and Dr. Ruvkun received the 2012 Dr. Paul Janssen Award for Biomedical Research for their contributions. MicroRNA's and Disease States The main function of microRNA’s is to translationally inhibit an mRNA transcript. MicroRNA’s are very small endogenous, non-coding, oligonucleotides. They are implicated in regulating a broad range of biological processes. They serve to bind via base complementarity rules to coding mRNA transcripts (which display the ‘sense’ sequence) either by inhibiting them or increasing their likelihood of degradation (most anti-sense therapeutics induce RNAase H cleavage, essentially a non-specific endonuclease). MicroRNA’s can drive gene expression and ultimately changes in concentration of a particular protein of interest. These are molecules which are involved in important regulation of biological processes, and the potential of using these molecules as targets for treatment is becoming ever more apparent. Theoretically, microRNA’s can target a variety of molecules composed of a natural RNA framework. Oligonucleotides can be designed to target and inhibit miRNA’s; in some instances, miRNA’s can actually act as translational activators. The premise behind which technology is useful is entirely dependent on the mechanism of action in relation to the disease. If an miRNA of interest is overexpressed in a particular disease state, targeting that miRNA via exogenous delivery of synthetic antagonistic molecules (anti-miRNA’s) may prove to be a powerful therapeutic approach. Different anti-miRNA’s are defined by specific modifications which allow higher affinity and functionality. Overcoming Hurdles Delivering these molecules efficiently and effectively (high affinity to their RNA target) remains a challenge, but recent advances in the laboratory suggest that RNA-based therapeutics may become an effective form of treatment. In 2002, the medical community reported a connection between microRNA and cancer; as research ensued in regards to their regulatory roles, microRNA's were found to be a useful tool for developing biomarkers. Mir-132, for example, was shown to be elevated in patients with osteoarthritis. Other specific miRNA’s were shown to be elevated in breast cancer. In fact, previously reported data has shown 29 distinct miRNA’s differentially expressed between normal mammary tissue and primary breast carcinomas. A little searching reveals a host of other documented aberrant expression profiles in relation to different diseases. Theoretical Evolves Into Reality Breast cancer is the most common form of cancer in women within the U.S. Approximately 1 woman out of 8 is at risk to develop breast cancer during the course of her lifetime. Breast cancer rates are substantially high, even in females who have no familial history. MiRNA’s can act as either tumor suppressors or oncogenes, depending on which biological pathway they regulate. Experimental data has shown that small miRNA’s which act as oncogenes (called ‘oncomirs’) do exist. Synthetic oligonucleotides which are capable of binding and thus inhibiting these molecules can represent a feasible and novel therapeutic approach to downregulate oncogene expression. Particular microRNA’s, such as mir-10b, have been shown to be highly expressed only in metastatic breast cancer cells, both in mice and human models. Collaboration between research labs at MIT and Sloan Kettering detailed how the miRNA mir-10b plays a role in metastasis. Results showed that overexpression of mir-10b in otherwise non-metastatic cells initiates cell migration and invasion. A transcription factor (called Twist) causes increases expression of mir-10b, and in turn this miRNA inhibits the translation of a homeobox gene. Consequently, this increases the expression of a documented pro-metastatic gene, RHOC. Later research published showed that therapeutic treatment with antagonistic (chemically modified) mir-10b oligonucleotides prevented metastasis. Interestingly, this has also shown that antagomirs were capable of being efficiently delivered after being chemically modified. It was previously reported that unmodified anti-sense oligonucleotides were quickly degraded after administration. The authors noted that a single miRNA targets many transcripts, and thus this entire network needs to be explored. From the Lab to Market Myocardial Ischemia, typically due to atherosclerosis of the coronary arteries, affects approximately 5 million people nationwide and is the leading cause of death in the United States. An article published in Circulation by Division of Cardiology at Stanford University in 2010 used a mouse model to show over-expression of mir-210 in cardiac muscle cells. This specific microRNA has been previously shown to upregulate the development of new blood vessels and prevent apoptosis. After undergoing intramyocardial injections with non-viral vectors containing a mir-210 precursor, improvements in left-ventricular pump function were seen after 8 weeks of treatment. In addition, histological analyses showed decreased cell death and increased neovascularization. This study provides a model, based on a particular microRNA, for a novel therapeutic approach in regards to treating myocardial infarctions. ********** Each year, several million people are affected by Hepatitis C, with the highest infection rates in African and eastern Mediterranean regions. The Hepatitis C Virus (HCV) primarily effects the liver. Current treatments for HCV, involving Pegasys and Ribavirin, are able to sustain clearance in 50% of patients, but the side effects are far too great. Previously, it has been shown that miR-122 (a highly miRNA expressed in the liver) binds to two required sites (in a non-coding region) of the HCV genome, resulting in the up regulation and subsequent accumulation of HCV. Research published in 2010 out of the National Primate Research Center in San Antonio, Texas, details a promising approach utilizing miR-122 as a target. Researchers utilized ‘SPC3649’, an LNA antagonist (provided by Santaris A/S) to miR-122 in chronically infected chimpanzees. Subjects were treated with intravenous injections every week for 12 weeks, with a subsequent treatment-free duration of 17 weeks; the study consisted of a high dose group and a low dose group. Data shows SPC3649 decreased HCV levels in the serum and liver in the high dose group after 2 weeks, and the low dose group experienced decreased HC levels in one animal and fluctuations in another. Northern blotting detected a shifted band depicting miR-122 in a bound state with SPC3649. Liver biopsies displayed improvement of HCV-induced liver disease. Recent phase-1 clinical trials were undertaken by Santaris with ‘SPC3649’ in healthy patients to assess toxicity. It is the first drug mRNA antagonist to enter clinical trials and it showed a well-tolerated profile with dose-dependent pharmacology. ********** The technology is now moving from the lab and into the clinical development space. Companies leading the miRNA industry include Sarepta, Mirna Therapeutics, Regulus Therapeutics, Miragen Therapeutics and Moderna Messenger Therapeutics. At one point, Big Pharma relied entirely on its own assets and intellectual property to further their research endeavors and/or interests. Now, Big Pharma instead has increased strategic relationships with these and other sector-focused companies to develop technology. These alliances allow for both parties to benefit from each other. Miragen, for example, has formed a strategic alliance with Santaris so it can utilize the pharma giant's proprietary LNA (Locked Nucleic Acid) drug delivery technology. LNA technology increases the drug’s affinity to the RNA target. Anti-sense pharmacology is not only limited by possibly toxicity, but also the chance of uptake. As mentioned before, they are quickly degraded if they are composed of Ribose or Deoxy-Ribose sugar-phosphate backbone. Chemical modifications can create an escape from degradation and create a more stable bond with the transcript. Toxicity can become an issue with this approach, however, and thus some technologies use a vector delivery mechanism which involves a lot of variables and thus low efficacy in most cases. The future of microRNA is vast, and the potential to meet significant medical needs are profound. As the technology moves out of the benchtop and into the clinical space, it seems both big pharma and specialized biotech companies are actively working to turn this technology into a reality. The next step will be larger scale studies with tangible, monetizing indications, and in turn, the development of a regulatory environment to support this innovation.