MicroRNAs: A Gene Silencing Mechanism with Therapeutic Implications

Resources

Companies/institutes dedicated to RNA therapies

Regulus Therapeutics

Mirna Therapeutics

miRagen Therapeutics

Institute for RNA Medicine, Harvard University

Keynote Presentation

David Bartel

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

Paul Grint

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.

Regulus Therapeutics clinical pipeline

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

Joana Vidigal

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.

Sponsors

Bronze Sponsor

Mirna Therapeutics

Grant Support

This program was supported by an educational grant from Bristol-Myers Squibb.

The Biochemical Pharmacology Discussion Group is proudly supported by:

Premiere Supporter

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.

miRNAs are transcribed in the nucleus and exported into the cytoplasm where they bind to proteins to form the RISC. miRNAs then bind to target sequences within mRNAs and inhibit their expression by promoting mRNA degradation and reducing translation efficiency. (Image courtesy of Frank Slack)

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.

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.

During the embryonic stage of zebrafish development, mRNA tail length correlates with translation efficiency—transcripts with longer polyA tails are translated more efficiently (2 hours post fertilization (hpf) and 4 hpf). However, later in development (6 hpf), as the zygote begins to transcribe its own RNA, this correlation is lost, and mRNA tail length does not affect translation. (Image courtesy of David Bartel)

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.

Highlights

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 mouse model of liver cancer, MRX34 improved survival compared to sorafenib, the current standard of care (left). MRX34 also reduced levels alpha-fetoprotein (AFP), a serum tumor marker (right). (Image courtesy of David Hong)

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.

Modular degradable dendrimers form nanoparticles that can deliver miRNAs to tumor cells within the liver. The core (blue) and periphery (red) functional groups can be modified for effective drug delivery. The degradable linkers (green) ensure that the particle breaks down in the cell, minimizing toxicity. (Image courtesy of Daniel Siegwart)

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.

Highlights

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.

MTP inhibitors such as lomitapide lower cholesterol levels by inhibiting lipoprotein secretion. This leads to a buildup of fatty acids in the liver and eventually liver steatosis. miR-30c also reduces cholesterol levels by inhibiting lipoprotein secretion by downregulating MTP, however it also inhibits proteins involved in lipid synthesis, thus preventing steatosis. (Image courtesy of M. Mahmood Hussain)

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.

RG-101 incorporates three RNA technologies to improve drug potency and targeting. The addition of the carbohydrate N-acetylgalactosamine (GalNAc) targets the drug to liver cells. A cleavable linker allows the drug and GalNAc to be cleaved once inside the cell. Generation 2.5 chemistry improves the drug's potency. (Image courtesy of Paul Grint)

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.

Highlights

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.

The miR-17-92 cluster contains six miRNAs belonging to four families (top, colored yellow, green, blue, and pink). Some phenotypes, such as defects in the heart and lungs, are associated with deletion of all four miRNA families (left). Other phenotypes, such as defects in the vertebrae, can be recapitulated by deleting one or two of the families (right). (Image courtesy of Joana Vidigal)

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.

Highlights

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?