MicroRNAs: A Gene Silencing Mechanism with Therapeutic Implications
Posted December 12, 2016
MicroRNAs (miRNAs) are short, noncoding RNA molecules that repress the translation of target mRNAs. Because of their roles in many important cellular processes, miRNAs are attractive candidates as drug targets, or as drugs themselves. Several miRNA therapies have recently made their way into clinical trials for cancer and hepatitis C virus (HCV) infection, while preclinical research is underway in a variety of other diseases.
On July 13, 2016, clinicians and basic researchers from academia and industry convened at the New York Academy of Sciences for the MicroRNAs: A Gene Silencing Mechanism with Therapeutic Implications symposium. Presenters discussed preclinical and clinical data for miRNA-based therapy as well as new insights in the basic functions of miRNAs. The meeting was presented by the Biochemical Pharmacology Discussion Group and the New York Academy of Sciences.
Use the tabs above to find a meeting report and multimedia from this event.
Presentations available from:
David Bartel (Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute, MIT)
Frank Slack (Beth Israel Deaconess Medical Center, Harvard Medical School)
Joana Vidigal (Memorial Sloan Kettering Cancer Center)
M. Mahmood Hussain (SUNY Downstate Medical Center)
Hin Hark Gan (New York University)
David S. Hong (The University of Texas MD Anderson Cancer Center)
The Biochemical Pharmacology Discussion Group is proudly supported by:
How to cite this eBriefing
The New York Academy of Sciences. MicroRNAs: A Gene Silencing Mechanism with Therapeutic Implications. Academy eBriefings. 2016. Available at: www.nyas.org/miRNA-eB
Companies/institutes dedicated to RNA therapies
Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009;19:92-105.
Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005;120:15-20.
Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell. 2007;27:91-105.
Garcia DM, Baek D, Shin C, Bell GW, Grimson A, Bartel DP. Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNAs. Nat Struct Mol Biol. 2011;18:1139-1146.
Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. Elife. 2015;4. doi:10.7554/eLife.05005.
Shin C, Nam JW, Farh KK, Chiang HR, Shkumatava A, Bartel DP. Expanding the microRNA targeting code: functional sites with centered pairing. Mol Cell. 2010;38:789-802.
Subtelny AO, Eichhorn SW, Chen GR, Sive H, Bartel DP. Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature. 2014;508:66-71.
microRNAs in Cancer
David S. Hong
Cortez MA, Ivan C, Valdecanas D, et al. PDL1 regulation by p53 via miR-34. J Natl Cancer Inst. 2015;108:pii djv303.
Hong DS, Kang Y-K, Brenner AJ. MRX34, a liposomal miR-34 mimic, in patients with advanced solid tumors: final dose-escalation results from a first-in-human phase I trial of microRNA therapy. J Clin Oncol. 2016;34(Suppl):abstract 2508.
Wang Xi, Li J, Dong K, et al. Tumor suppressor miR-34a targets PD-L1 and functions as a potential immunotherapeutic target in acute myeloid leukemia. Cell Signal. 2015;27:443-452.
Daniel J. Siegwart
Love KT, Mahon KP, Levins CG, et al. Lipid-like materials for low-dose, in vivo gene silencing. Proc Natl Acad Sci USA. 2010;107:1864-1869.
Wu L, Nguyen LH, Zhou K. Precise let-7 expression levels balance organ regeneration against tumor suppression. Elife. 2015;4:e09431.
Zhou K, Nguyen LH, Miller JB, et al. Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc Natl Acad Sci USA. 2016;113:520-525.
Frank J. Slack
Kasinski AL, Slack FS. MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy. Nat Rev Cancer. 2011;11(12):849-864.
Kasinski AL, Kelnar K, Stahlhut C, et al. A combinatorial microRNA therapeutics approach to suppressing non-small cell lung cancer. Oncogene. 2015;34(27):3547-3455.
MicroRNAs in Heart Disease and HCV
Horvath G, Papatheodoridis G, Fabri M, et al. RG-101 in combination with 4 weeks of oral direct acting antiviral therapy achieves high virologic response rates in treatment naïve genotype 1 and 4 chronic hepatitis C patients: interim results from a randomized, multi-center, phase 2 study. The International Liver Congress (EASL). April 13 17, 2016; Barcelona, Spain. Abstract GS08.
PRNewswire. La Jolla, CA. Regulus reports clinical hold of RG-101. [press release]. June 27, 2016.
M. Mahmood Hussain
Irani S, Pan X, Peck BC, Iqbal J, Sethupathy P, Hussain MM. MicroRNA-30c mimic mitigates hypercholesterolemia and atherosclerosis in mice. J Biol Chem. 2016. Epub ahead of print.
Soh J, Hussain MM. Supplementary site interactions are critical for the regulation of microsomal triglyceride transfer protein by microRNA-30c. Nutr Metab (Lond). 2013;10:56.
Soh J, Iqbal J, Queiroz J, Fernandez-Hernando C, Hussain MM. MicroRNA-30c reduces hyperlipidemia and atherosclerosis in mice by decreasing lipid synthesis and lipoprotein secretion. Nat Med. 2013;19:892-900.
Basic Studies on miRNA Function
Han YC, Vidigal JA, Mu P, et al. An allelic series of miR-17 ~ 92-mutant mice uncovers functional specialization and cooperation among members of a microRNA polycistron. Nat Genet. 2015;47:766-775.
Vidigal JA, Ventura A. The biological functions of miRNAs: lessons from in vivo studies. Trends Cell Biol. 2015;25:137-147.
Hin Hark Gan
Gan HH, Gunsalus KC. Assembly and analysis of eukaryotic Argonaute-RNA complexes in microRNA-target recognition. Nucleic Acids Res. 2015;43:9613-9625.
Gan HH, Gunsalus KC. Tertiary structure-based analysis of microRNA-target interactions. RNA. 2013;19:539-551.
Lynn M. Abell, PhD
Lynn Abell is a senior principal scientist for Bristol-Myers Squibb, where she is responsible for leading the enzymology group.
Andreas G. Bader, PhD
Andreas Bader joined Mirna Therapeutics in December 2007 and has served as director of analytical and external research since January 2012. Previously, he served as the project lead of the microRNA therapeutics program at Asuragen. Prior to joining Asuragen, Bader held a faculty position at The Scripps Research Institute, La Jolla, California, where he also completed his postdoctoral training, focusing on molecular mechanisms of cancer and the identification of novel cancer drug targets. Bader received his MSc in biology and his PhD in biochemistry from the University of Innsbruck, Austria.
David Brown, PhD
David Brown serves as vice president of preclinical pharmacology at Mirna Therapeutics and has worked with the company since its inception in 2008. Previously, Brown was the director of discovery at Asuragen, Inc., where he oversaw efforts to identify diagnostic and therapeutic applications for microRNAs. Prior to joining Asuragen, Brown worked for ten years as a senior scientist and director of research and development at Ambion, Inc. Brown received his PhD in molecular biology from the University of Colorado.
Paul Lammers, MD
Paul Lammers joined Mirna Therapeutics in 2009 as its president, CEO, and board director. Prior to joining Mirna Therapeutics, Lammers served as president of Repros Therapeutics in The Woodlands, Texas, after having served for six years as the chief medical officer for EMD Serono Inc., a division of German pharmaceutical company, Merck KgA. He began his career with Dutch pharmaceutical company, Organon, spending eight years in the commercial and clinical operations in Europe and the US. He also served four years as senior vice president of clinical and regulatory affairs at Zonagen in The Woodlands, Texas. Lammers obtained his medical and masters of science degrees from Radboud University in Nijmegen, The Netherlands, and moved with his family to the US in 1992.
George Zavoico, PhD
George Zavoico is a senior equity healthcare analyst at JonesTrading Institutional Services, where he focuses principally on biotechnology, biopharmaceutical, specialty pharmaceutical, and molecular diagnostics companies.
In 2009, Zavoico was hired as the first equity analyst at New York-based investment bank MLV & Co., where he helped establish its healthcare research team. Prior to that, Zavoico was an equity research analyst in the healthcare sector at Westport Capital Markets and Cantor Fitzgerald. Previously, Zavoico established his own consulting company serving the biotech and pharmaceutical industries. Zavoico began his career as a senior research scientist at Bristol-Myers Squibb Co. Zavoico has a bachelor's degree in biology from St. Lawrence University and a PhD in physiology from the University of Virginia. He held post-doctoral fellowships at the University of Connecticut School of Medicine and Harvard Medical School / Brigham & Women's Hospital. He has published more than 30 papers in peer-reviewed journals and has coauthored four book chapters.
Sonya Dougal, PhD
The New York Academy of Sciences
Caitlin McOmish, PhD
The New York Academy of Sciences
David Bartel, PhD
Since receiving his PhD from Harvard in 1993, David Bartel has headed a lab at the Whitehead Institute for Biomedical Research, where he is also an investigator of the Howard Hughes Medical Institute and a professor of biology at MIT. His lab initially studied the ability of RNA to catalyze reactions and more recently has focused on RNAs that regulate gene expression. Over the past 16 years, his lab has helped define microRNAs and other types of small regulatory RNAs and has contributed to the understanding of their genomics, biogenesis and regulatory targets, as well as the molecular and biological consequences of their actions in animals, plants and fungi.
Paul Grint, MD
Paul Grint joined Regulus in 2014 as chief medical officer and was appointed president and chief executive officer in 2015. Grint has over two decades of experience in biologics and small molecule development, including the successful commercialization of numerous commercial products in oncology, anti-infectives and immunology in both domestic and international markets. Prior to joining Regulus, Grint was president of Cerexa, Inc., where he was responsible for the oversight of anti-infective product development. Prior to that, Grint served as senior vice president of research at Forest Research Institute, Inc., chief medical officer at Kalypsys, Inc., and senior vice president and chief medical officer at Zephyr Sciences, Inc., as well as at Pfizer Inc., IDEC Pharmaceuticals Corporation, and Schering-Plough Corporation. Grint received his bachelor's from St. Mary's Hospital in London and his MD from St. Bartholomew's Hospital Medical College at the University of London. Grint is a fellow of the Royal College of Pathologists, and the author or co-author of over 50 scientific publications.
David S. Hong, MD
David Hong joined the University of Texas MD Anderson Cancer Center in 2005. He received his medical degree from Albert Einstein College of Medicine, Yeshiva University, in New York and served as an intern and resident at Thomas Jefferson University in Philadelphia. Hong completed a fellowship in medical oncology at MD Anderson, where he was appointed chief medical oncology fellow. His research interests include phase I studies, drug development, the molecular basis for cancer clinical interest therapy, biologic molecules, and novel protocol designs. Hong has received numerous awards including the Amgen Young Investigator Award, the Jesse H. Jones Award and the Goodwin Funding Award.
Daniel J. Siegwart, PhD
Daniel Siegwart is an assistant professor in the Simmons Comprehensive Cancer Center and biochemistry department at the University of Texas Southwestern Medical Center (UTSW). He received a BS in biochemistry from Lehigh University, and a PhD in chemistry from Carnegie Mellon University (CMU). He has received the Joseph A. Solomon Memorial Fellowship in Chemistry at CMU and was a National Science Foundation East Asia and Pacific Summer Institutes Fellow at the University of Tokyo. Siegwart then completed a National Institute of Health sponsored postdoctoral fellowship at MIT. Siegwart began his independent research career in 2012 at UTSW. The Siegwart Research Group's long-term goals are to develop new materials for therapeutic nucleic acid delivery, develop new polymers to deliver chemotherapeutic drugs to hypovascular tumors, develop theranostic "turn on" probes, and to globally understand how the physical and chemical properties of materials affect interactions with biological systems in vitro and in vivo in the context of improving cancer therapies.
Frank Slack is director of the institute for RNA medicine at Beth Israel Deaconess Medical Center, where he is a professor of pathology.
Slack received his BSc from the University of Cape Town in South Africa, before completing his PhD in molecular biology at Tufts University School of Medicine. He started work on microRNAs as a postdoctoral fellow at Harvard Medical School, where he co-discovered the second known microRNA, let-7, and the first human microRNA. Subsequently, he moved to Yale University, where he was a program leader in the Yale Cancer Center and director of the Yale Center for RNA Science and Medicine. There he discovered that microRNAs regulate key human oncogenes and have the potential to act as therapeutics. He also demonstrated the first role for a microRNA in the aging process.
Slack was an Ellison Medical Foundation Senior Scholar and received the 2014 Heath Memorial Award from MD Anderson Cancer Center.
Joana Vidigal received her PhD from the Free University of Berlin in 2011, where she developed an inducible RNAi platform to study mouse embryogenesis. Since then, as a research fellow in the lab of Andrea Ventura at Memorial Sloan Kettering Cancer Center, she has been focused on understanding the role of noncoding elements—and in particular miRNAs—in controlling gene expression, and how their deregulation can contribute to disease. Using an allelic series of genetically engineered mouse models, Vidigal helped define the functional contributions of individual components of the oncogenic miR-17~92 cluster to the regulation of mammalian development and tumor progression. She is currently establishing CRISPR-Cas9 tools to functionally query the noncoding genome in a high-throughput manner. Vidigal is a recipient of the 2016 Memorial Sloan Kettering Postdoctoral Research Award.
M. Mahmood Hussain
SUNY Downstate Medical Center
Hin Hark Gan
New York University
Jennifer Cable lives in New York City, where she experiments with different outlets to communicate science. She enjoys bringing science to scientists and nonscientists alike. She writes for Nature Structural and Molecular Biology, Bitesize Bio, Under the Microscope, and the Nature New York blog. She received a PhD from the University of North Carolina at Chapel Hill for her research in investigating the structure/function relationship of proteins.
This program was supported by an educational grant from Bristol-Myers Squibb.
The Biochemical Pharmacology Discussion Group is proudly supported by:
MicroRNAs (miRNAs) are short noncoding RNA molecules about 22 nucleotides long that bind to target sequences typically within the untranslated region of messenger RNAs (mRNAs) and downregulate their expression. Many human genes, and possibly the majority of them, are regulated by miRNAs, adding an additional layer of gene control to fine-tune protein expression.
miRNAs are involved in almost all major cell functions, including differentiation, motility, and survival. Their ubiquity and importance make them attractive drug targets for many diseases. Since the discovery of miRNAs in 1993, miRNA therapies have quickly found their way into clinical trials for cancer and hepatitis C virus (HCV) infection. In addition, preclinical work is underway in heart disease, neurological disorders, and metabolic diseases. Traditional small molecule drugs often bind to pockets in protein targets. However, many proteins do not have easily accessible binding pockets. miRNAs, and other RNA-based therapies, offer the promise of targeting these undruggable targets, including many genes important in cancer, such as p53, KRAS, and BRCA.
There are several approaches for miRNA-based therapeutics. miRNAs can act as drugs themselves to downregulate genes. Conversely, anti-miRNA drugs can be used to inhibit miRNAs to increase the levels of their targets. In addition, small molecule drugs can target proteins involved in miRNA synthesis or miRNA targets. During the symposium, the speakers focused on the first two strategies—miRNAs as therapeutics and as therapeutic targets.
Several speakers talked about the use of miRNA-based therapies in cancer. Frank J. Slack, Harvard University, discussed preclinical data on miRNA therapies in lymphoma, breast cancer, and lung cancer. David Hong, University of Texas MD Anderson Cancer Center, presented data on a clinical trial for MRX34, an experimental miRNA mimic developed by Mirna Therapeutics for patients with solid tumors. Daniel J. Siegwart, University of Texas Southwestern Medical Center, discussed his work on developing new technologies to deliver miRNA therapies to target tissues and their application in liver cancer.
While oncology has been the focus for much of the miRNA research, miRNA therapies hold promise for many other diseases. Paul Grint, Regulus Therapeutics, discussed the clinical development of RG-101, an experimental miRNA-based therapy for HCV infection, while M. Mahmood Hussain, SUNY Downstate Medical Center, presented his research testing miRNA therapies for heart disease in mouse models.
Other speakers presented their research on understanding the function of miRNAs. Joana Vidigal, Memorial Sloan Kettering Cancer Center, presented her work on dissecting the role of individual miRNAs clustered within the genome. Hin Hark Gan, New York University, discussed his work on characterizing how single nucleotide polymorphisms (SNPs) within miRNA targets affect miRNA binding.
David P. Bartel
Massachusetts Institute of Technology
Predicting how miRNAs recognize their targets
In his keynote, David Bartel of the Massachusetts Institute of Technology provided an overview of the current understanding of how miRNAs repress mRNA translation and emphasizing his research on predicting how effective miRNAs are at repressing their targets.
One of the primary factors dictating miRNA efficiency is the number of targets sites within an individual mRNA transcript. Each transcript may have several miRNA target sites that have small effects on expression individually, but can dramatically repress expression when engaged together.
Bartel also looked at how the sequence of the mRNA target site affects miRNA repression. mRNA targets typically base pair with nucleotides 2–7 in the miRNA, known as the seed sequence. While these are the most important bases for binding to mRNA, Bartel showed that bases flanking the seeds sequence can affect the efficiency of repression and can compensate for less than perfect complementarity. Bartel also showed that many mRNA target sites identified via crosslinking experiments are unlikely to be functional and do not downregulate mRNA expression.
Finally, the accessibility of the mRNA target site is also important. Some sites may be engaged in secondary structure and inaccessible for binding to miRNA.
Based on these criteria, Bartel has developed a model to identify miRNA binding sites within mRNA and to estimate the efficiency of miRNA repression. Researchers interested in whether an mRNA is likely to be repressed by miRNA can view these predictions at TargetScan.org.
miRNAs and translational repression
Bartel also described his work in better understanding the mechanisms by which miRNAs repress expression. While miRNAs primarily repress mRNAs by destabilizing the transcript, a portion of their effect is mediated by changes in translation. He tested whether shortening of the mRNA polyA tail, a string of adenine nucleotides found at the 3′ end of mRNA, by the deadenylase complex in the RNA-induced silencing complex (RISC) reduces translation efficiency.
It has been well known that there is a direct correlation between length of polyA tails and increased translation efficiency in oocytes and early embryos. However, after measuring the tail lengths of thousands of genes in different cell types and organisms, Bartel found that while short tail lengths do correspond to low translational efficiency in embryos, this correlation does not exist during later periods in development. Bartel is interested in further exploring why tail length appears to be important during early stages of development but not later.
Frank J. Slack
Beth Israel Deaconess Medical Center, Harvard Medical School
Daniel J. Siegwart
University of Texas Southwestern Medical Center
David S. Hong
University of Texas MD Anderson Cancer Center
miRNAs are frequently dysregulated in cancer.
miR-155 is a promising target for lymphoma treatment based on mouse studies, and an anti-miR-155 is currently in early clinical testing.
A phase 1 trial of MRX34, a miR-34 mimic, has shown some promising results, but due to safety concerns, the drug is on clinical hold.
Nanoparticles based on degradable dendrimers have been shown to target miRNA therapies to tumors in mouse models of cancer.
The potential for miRNA therapies in cancer treatment
Dysregulation of miRNAs is often implicated in cancer—miRNAs have been shown to regulate many important cancer genes, and genes that encode miRNAs are frequently located in areas of the genomes that are deleted or amplified in cancer patients and mis-expressed in tumors. In addition, many cancer patients have mutations in enzymes involved in miRNA processing, such as Drosha, Exportin, and Dicer.
Because miRNAs can regulate oncogenes and tumor suppressors, the therapeutic strategy depends on what is driving the cancer. In tumors with an activated oncogene, upregulating an miRNA that controls that oncogene can suppress its activity. Conversely, an amplified miRNA could be causing excess downregulation of a tumor suppressor gene. Targeting that miRNA with an anti-miRNA could help to normalize expression of that gene.
miRNA therapies in mouse models of cancer
Frank J. Slack of Beth Israel Deaconess Medical Center, Harvard Medical School described preclinical studies of miRNA therapies in cancer. In mice, overexpression of miR-155, a microRNA expressed in hematopoietic cells, induces lymphoma while turning off expression causes tumors to regress. Slack showed a nucleotide-based anti-miR-155 drug that binds to and inhibits miR-155 could cure lymphoma in mice. To ensure that the drug targets tumor cells while ignoring normal cells, Slack's group fused to a peptide that preferentially inserts into cells under acidic conditions, such as the tumor environment.
Slack cautioned against touting anti-miR-155 as a lymphoma cure for humans. In the mouse model, the precise cause of the lymphoma was known, whereas in human disease, there may be many unknown factors. Nonetheless, anti-miR-155 does represent a promising strategy. In work unrelated to Slack's, miRagen Therapeutics has started a phase 1 clinical trial of an anti-miR-155 drug in patients with lymphoma. The trial is currently enrolling patients.
Slack also discussed a collaboration with Mirna Therapeutics on miR-34 and the let-7 miRNAs in breast and lung cancer. These miRNAs repress major oncogenes, including RAS, MYC, and BCL-2, and are often deleted in breast and lung cancer patients.
In a mouse model of highly aggressive non-small cell lung cancer (NSCLC), administering either miR-34 or a let-7 miRNA, caused tumor reduction and increased survival, and a combination of the two had an even larger effect. The drugs were delivered to the lung via nanoparticles called Smarticles, which are small liposomes that release the miRNA in the acidic environment of tumors. In breast cancer cell lines, adding miR-34 induced senescence and stopped cell replication. In mice with breast cancer, addition of miR-34 slowed tumor growth and induced apoptosis. No toxicity was observed in mouse models, however Slack cautioned that the mice were treated for a short time, whereas cancer patients would receive long-term treatment.
miRNAs as immune modulators in cancer
David S. Hong of the University of Texas MD Anderson Cancer Center also discussed the potential for miR-34 therapies in cancer. He presented recent clinical data on MRX34, an miR-34 mimic encapsulated in Smarticles developed by Mirna Therapeutics that is being studied in patients with solid tumors.
miR-34 is a tumor suppressor—high levels of miR-34 correlate with longer survival in patients with cancer, and in preclinical studies, overexpression of MRX34 increased survival in a mouse model of liver cancer. In addition to downregulating several oncogenes, also affects PDL1, a cell receptor that is overexpressed in cancer and allows tumors to evade the immune system. PDL1 is a target for several cancer immunotherapies in development. Hong showed data that suggests that the efficacy of MRX34 in solid tumors is at least partially due to its effects on the immune system through PDL1.
In a Phase 1 trial presented in 2016 at the American Society of Clinical Oncology conference, MRX34 was given to patients with solid tumors. As expected, MRX34 downregulated several miR-34 targets. Of the 107 patients treated, 4 experienced greater than 30% reduction in tumor size. One of these patients experienced pseudoprogression, a phenomenon that occurs in some patients taking immunotherapy in which the tumor initially gets larger, presumably due to infiltrating immune cells, before shrinking. In addition, 15 patients had stable disease, in which the tumor size either stays the same or does not decrease more than 30%. With regard to safety, most patients experienced side effects typical of immune therapy, such as fever and fatigue. While many of these side effects were not serious, there were two deaths, one due to pneumonitis/colitis, a side effect indicative of immune system activation. As of the publication of this report, Mirna Therapeutics has voluntarily stopped this Phase 1 trial due to immune-related safety concerns and has decided not to initiate a trial in melanoma patients, which was planned to start in late 2016. As of press time, according to the company, it is still considering whether to continue development of MRX34.
Designing carrier systems for miRNAs
Daniel J. Siegwart of University of Texas Southwestern Medical Center described his work in designing an efficient nanoparticle carrier for miRNAs. The goal of any drug delivery mechanism is to deliver the drug to the cells of interest while avoiding normal cells to limit off-target effects. Siegwart has developed nanoparticles made of dendrimers, synthetic branched polymers that can be customized with various functional groups. He attached positively charged amine groups that bind to negatively charged RNA molecules that would be released in the neutral pH environment inside the cell. Degradable linkers allow the particle to be broken down within the cell to mitigate toxic effects.
Siegwart tested various functional groups and polymer lengths to determine which chemical and physical properties were amenable to cellular uptake. He then took some of the lead compounds that inserted into liver cells, without any miRNA drug, and tested their toxicity in mice. In healthy mice, the nanoparticles had no toxic effect. However, in a mouse model of liver cancer, some of the particles caused drastic weight loss and death. One of the particles, however, was nontoxic and localized to liver tumor cells. Siegwart then added the miRNA let-7g to this nanoparticle and showed that it localized to tumor cells in the liver and dramatically increased survival in mice with liver cancer. Siegwart is now using the nanoparticle toolbox he developed to test other miRNAs and tumor types in mouse experiments.
M. Mahmood Hussain
SUNY Downstate Medical Center
miRNA therapies have shown promise in mouse models for reducing plasma cholesterol levels and preventing atherosclerosis.
RG-101, an miRNA antagonist in clinical studies for the treatment of HCV, may help reduce treatment duration, although safety issues have placed the trial program on hold.
miRNA therapies for heart disease
High cholesterol is a major risk factor for cardiovascular disease. Cholesterol is carried through the bloodstream in lipoproteins—particles made up of lipids and protein. There are two major types of lipoprotein, low density lipoproteins or LDL, also known as "bad cholesterol", which is the primary culprit for atherosclerosis and heart disease, and high density lipoprotein or HDL, also known as "good cholesterol", which has a protective effect against heart disease.
There are two major strategies to control high LDL levels—increase lipoprotein clearance or turn off cholesterol synthesis. The most common cholesterol therapies, statins and PCSK9 inhibitors, increase lipoprotein clearance by upregulating the LDL receptor, which removes LDL from the blood. However, these drugs are not as effective in patients with high cholesterol due to homozygous familial hypercholesterolemia (HoFH), who have defective LDL receptors. For these patients, adjunct therapies that further reduce lipid synthesis can be beneficial. Two drugs target high cholesterol via this mechanism—lomitapide, which inhibits microsomal triglyceride transfer protein (MTP), and mipomersen, which reduces the production of apolipoprotein B (ApoB). Both MTP and ApoB are involved in lipid secretion and assembly. While these drugs reduce lipid levels in the blood, the lipids are not cleared by the body and instead remain in the liver, leading to fatty liver and steatosis.
M. Mahmood Hussain of SUNY Downstate Medical Center described his work investigating whether miRNAs could reduce plasma lipid levels while avoiding the fatty liver side effects associated with lomitapide and mipomersen. Hussain used the program TargetScan, described earlier by David Bartel, to identify microRNAs that regulate MTP. One of the hits was miR-30c. Injecting miR-30c into mice reduced plasma cholesterol levels and reduced the number of plaques in mice with atherosclerosis. miR-30c also affected proteins involved in lipid synthesis and did not lead to excess accumulation of lipids in the liver.
Hussain is also investigating whether miRNA species can decrease LDL and increase HDL levels. By screening a microRNA library, Hussain observed that miR-1200 lowers apolipoprotein B, a major component of LDL, and increases apolipoprotein A1, a major component of HDL. He also found that miR-1200 had these effects in mice. In addition, miR-1200 also reduces fatty acid oxidation by decreasing the nuclear receptor co-repressor 1 (NCOR1). In total, these effects appear to reduce atherosclerosis in mice.
Clinical data for an anti-miRNA against HCV
Despite recent approvals and advancements in the treatment of HCV, the majority of infected patients around the world have not been treated, and the number of infected individuals is on the rise. Paul Grint of Regulus Therapeutics explained that there are several unmet needs in the HCV field that Regulus Therapeutics is hoping to address with miRNA therapies. One goal is to shorten the duration of therapy. Current drugs generally require 12 weeks of treatment, and previous attempts to reduce the time of treatment to 4 weeks have proven unsuccessful. The other goal is to provide treatment options for patients who cannot take the new oral drugs, such as those with end-stage renal disease (ESRD).
Grint described the clinical development of RG-101, an anti-miRNA for HCV infection. RG-101 binds to and inhibits miR-122, a microRNA found in the liver that regulates the synthesis of cholesterol and fatty acids. During the life cycle of HCV, the virus uses miR-122 to protect its own RNA from degradation. Preventing HCV RNA from binding to miR-122 prevents viral replication.
RG-101 incorporates three advances in RNA technology. First, the anti-miR-122 RNA sequence is fused to an N-acetylgalactosamine (GalNAc) carbohydrate molecule that binds to a receptor on the surface of liver cells. Second, a cleavable linker between the miRNA and carbohydrate allows the drug to be released once inside the cell. Finally, the proprietary Generation 2.5 Chemistry increases the drug's potency. Grint showed that these modifications increased targeting to the liver by 20–30 fold compared to the naked anti-miRNA in both cell culture and mouse models.
In a phase 1 study, one injection of RG-101 produced drops in HCV levels similar to those seen in previous studies of oral, direct-acting antivirals (DAAs). While viral levels recovered after approximately one month in most patients, three have been free of HCV for over one year. Unlike some oral drugs, RG-101 was effective against all HCV genotypes.
Regulus Therapeutics conducted a phase 2 study of RG-101 to determine whether a combination of RG-101 and approved oral drugs could shorten treatment duration. Patients received an injection of RG-101 followed by four weeks of an approved oral drug, either Olysio, Daklinza or Harvoni, a combination of sofosbuvir and ledipasvir. All patients who received RG-101 with Harvoni were free from the virus 12 weeks after treatment, compared to 96% of patients receiving RG-101 with Olysio and 92% receiving RG-101 with Daklinza. This was the first study to demonstrate a competitive response rate with only 4 weeks of treatment.
Regulus is also pursuing a collaboration with GlaxoSmithKline and the latter company's investigational long-acting anti-HCV DAA GSK-175. The company hopes that a single injection of RG-101 and GSK-175 may provide a single visit cure for patients.
While the clinical efficacy of RG-101 appears promising, serious safety signals caused the US Food and Drug Administration (FDA) to place the drug on clinical hold in June 2016. This decision was based on the occurrence of severe jaundice in two patients—one in the phase 2 study and one in a phase 1 study of end-stage renal patients. The phase 1 and 2 trials have already been completed, and Regulus is continuing to monitor these patients for any safety concerns, as of press time.
Hin Hark Gan
New York University
Memorial Sloan Kettering Cancer Center
Many miRNAs are clustered within the genome and expressed as a single transcript.
Each member of an miRNA cluster may function independently or cooperate with other members of the cluster to produce a specific phenotype.
SNPs within mRNA target sequences can affect how strongly miRNAs bind to their targets and may have functional consequences.
Deciphering miRNA clusters
Many miRNAs are expressed as part of a cluster so that a single RNA transcript contains several miRNAs that are later processed into individual miRNAs. Joana Vidigal of Memorial Sloan Kettering Cancer Center described her work on the miR17-92 cluster, which contains six miRNAs belonging to four families. miR-17-92 plays key roles in mammalian development and disease. In mice, deleting both copies of miR-17-92 causes severe developmental defects and death shortly after birth. In humans, deletion of one copy of the cluster results in Feingold Syndrome, characterized by abnormalities in the fingers and toes, blockages in the digestive system, and small head and jaw. Conversely, the cluster is often overexpressed in many human tumors.
Vidigal's work focuses on determining how each miRNA family within the cluster contributes to the effects seen upon deletion of the entire cluster. For example, one phenotype seen upon deletion of the mi-17-92 cluster in mice is changes in the vertebrae pattern. Vidigal found that a single miRNA family in the cluster, known as miR-17, controlled this phenotype. Other phenotypes, such as defects in the heart and lungs, require deleting several families or the entire cluster.
Vidigal also discovered that the miR-19 family is responsible for the cluster's role in tumorigenesis. Deleting miR-19 induced tumor cell death mouse models of lymphoma and prostate cancer. Vidigal hypothesized that inhibiting miR-19 might be a successful therapeutic strategy in some cancers.
Now that Vidigal has teased out the function of each miRNA within the miR17-92 cluster, she is interested in determining the biological basis for these functions. She is currently looking at how expression profiles change when miRNAs within the clusters are deleted to determine the downstream targets.
The effect of SNPs on miRNA binding
Single-nucleotide polymorphisms (SNPs) are variants in single nucleotides throughout the genome that are often linked to disease. Hin Hark Gan of New York University described his work in determining whether SNPs located within the miRNA target sequences of mRNAs affect how the miRNA machinery recognizes those targets.
Gan identified 19 SNPs within miRNA targets that have known functions. Using computational methods, he predicted the structures of complexes of miRNAs and mRNA targets containing a range of SNP variants. He then docked these duplex structures onto RISC.
Gan showed that SNPs within miRNA targets can create changes in the miRNA/target duplex structure that can either reduce or increase the binding affinity of the duplex to the RISC complex. He hopes that these findings can shed light on the correlation between these SNPs and disease.
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David P. Bartel
Massachusetts Institute of Technology
Daniel J. Siegwart
University of Texas Southwestern Medical Center
Frank J. Slack
Beth Israel / Deaconess Medical Center, Harvard University
Memorial Sloan Kettering Cancer Center
The panelists are optimistic that miRNA therapies will soon be part of the standard of care for many diseases.
Drug delivery remains one of the most important considerations for miRNA therapies.
While current drug delivery technologies are probably adequate for clinical testing and use in patients, there is room for improvement in tissue targeting and cellular uptake.
To conclude the conference, several of the speakers took part in a panel discussion to share their thoughts on the current state and potential future of miRNA therapies.
The future of miRNA therapies
Frank J. Slack put forth an ambitious prediction that RNA-based therapies will be part of the standard of care for many diseases in response to a question about where he would like to see the miRNA field in the next five to ten years. He also explained that he envisions noncoding sequences of the genome playing a larger role in medical decisions to improve diagnostics and expand personalized medicine. Achieving these objectives will require improvements in bioinformatics and big data storage and processing.
The other panelists provided concrete advances that they would like to see in the field of miRNAs. David P. Bartel noted that there is still a lot to learn about which miRNA targets are responsible for particular phenotypes. While much work has been done on knocking out miRNAs and identifying their function, it's often not known which target is responsible. New technologies that enable researchers to mutate target sites can help elucidate the specific targets.
Joana Vidigal also stressed the need to better understand the role of miRNA targets in development and cellular biology. Although her talk presented an example of a miRNA that causes a significant effect when deleted, many miRNAs do not produce an obvious effect when deleted. One hypothesis that Vidigal would like to investigate is that these miRNAs are involved in stress response, and some phenotypes might only be observed under certain conditions. Daniel J. Siegwart brought up the need for improvements in drug delivery. Although the potency of miRNA therapies has increased, very few RNA molecules are actually taken up into the target cell. Increasing the rate of cellular uptake could reduce the dose of therapy required to achieve the desired effect and reduce the chance of side effects.
miRNA drug delivery systems
When asked whether the current delivery technologies for miRNA drugs were adequate, Siegwart responded that, although there is always room for improvement, current technologies are already good enough to bring miRNA therapies into the clinic.
Slack did note, however, that large pharmaceutical companies do not seem interested in developing new RNA delivery technologies and that smaller biotech companies are the bulk of the research is being done by.
David Hong explained that the choice of Smarticles as the delivery mechanism for MRX34 came down to several factors—the number of RNA molecules that could be encapsulated into the liposome, the efficiency of delivery of RNA to the tumors, and efficacy in in vivo models.
There was also interest among attendees about delivering multiple types of miRNA species in the same therapy. Bartel explained that while it is possible to deliver different miRNAs in one particle, there will be competition between the miRNAs unless you have an extended-release delivery mechanism. Furthermore, delivering too many miRNA molecules introduces the risk of saturating the cell's miRNA machinery and preventing endogenous miRNAs from functioning properly.
Finally, there was a question from an audience member about whether small molecule drugs could be used to target miRNAs. Slack explained that pharmaceutical companies are more comfortable working with small molecules than miRNA or anti-mRNA based drugs, and although there is some interest in developing small molecules that target miRNAs, the field is small. Furthermore, targeting the miRNA assembly machinery is difficult to do selectively since it processes many different miRNAs, but it may be possible to target specific RNA-binding proteins to block processing of specific miRNAs.
How do miRNAs affect translation efficiency?
How does the polyA switch occur, transitioning into the developmental phase when polyA tail length ceases to be important for translation efficiency?
Which mRNA targets of miRNAs are responsible for miRNA phenotypes?
How do miRNAs within a cluster of miRNAs cooperate to produce phenotypes?
Are there conditions, such as stress, that are required to observe the phenotype of certain miRNAs?
Could SNPs in miRNA targets that affect miRNA/mRNA binding explain the functional consequences of these SNPs in disease?
What are the best drug delivery approaches for miRNA therapies?
How will preclinical data, such as the testing of anti-miR-155 therapies in lymphoma patients, translate into human clinical trials?