March 15, 2011 (Vol. 31, No. 6)

Kathy Liszewski

Emerging Research Corroborates Potential for Broad Application Across Many Diseases

MicroRNAs (miRNAs) are master regulators involved in multiple physiological and pathological processes. These naturally occurring, noncoding, single-stranded RNAs (21–25 nucleotides long) base-pair with their target mRNA within the RISC (RNA-induced silencing complex). The latter is the same ribonucleoprotein machinery associated with siRNA-mediated gene silencing.

While siRNAs are double-stranded oligonucleotides that perfectly pair to degrade their target mRNA, miRNAs can pair perfectly or imperfectly. Perfect base pairing leads to the degradation of the mRNA (similar to siRNA), while imperfect complementarity inhibits translation. It’s been suggested that miRNAs regulate up to 50% of all mRNAs in the human genome.

Discoveries linking miRNAs to a number of diseases have helped propel the fast-paced growth of this young field. Release of the first miRNA-based diagnostic test in 2008 helped reduce its failure risk. Keeping pace with new applications and tackling current issues was a focus of Keystone’s “MicroRNAs and Human Disease” conference held last month. Researchers described technological advances as well as novel miRNA therapeutics such as for cardiovascular disease, cancer, and muscular dystrophy.

Some of the same strategies already developed for delivery of siRNA for RNA interference (RNAi) also are being applied to miRNA. “Both miRNAs and siRNAs need to be delivered into the target tissue or cell in order to activate the desired therapeutic effect,” Muthiah Manoharan, Ph.D., senior vp, drug discovery, Alnylam Pharmaceuticals, explained.

“Chemical modifications to provide drug-like properties to RNA molecules are used in the synthesis of both siRNAs and antimicroRNAs (antimiRs). In addition, the lipid nanoparticles (LNP) platform, which has proven to be effective with siRNAs, is also showing promise with miRNAs and miRNA mimics.”

According to Dr. Manoharan, Alnylam is utilizing second-generation LNPs to enhance delivery of siRNAs in several models. “We demonstrated an approximate 100-fold improvement in potency over first-generation formulations, and achieved an effective dose (ED50) at single-digit microgram per kilogram dose levels. Additionally, we’ve made progress in hepatocyte-targeted in vivo gene silencing by modifying the siRNAs.

“We conjugated an N-acetylgalactosamine moiety (GalNac) to siRNAs and achieved improved potency using subcutaneous delivery at low, clinically relevant doses. In the case of a GalNAc-conjugated siRNA targeting transthyretin (TTR), target gene silencing was achieved with an ED50 of approximately 5 mg/kg with a single subcutaneous injection. These results represent a greater than 30-fold improvement in target gene silencing with siRNA conjugates as compared with first-generation siRNA conjugates previously described.”

Alnylam Pharmaceuticals is using its lipid nanoparticles platform to enhance delivery of siRNAs in several models. It reportedly delivers a greater than 30-fold improvement in target gene silencing.

Targeting Cancer

The involvement of miRNAs in cancer research emerged from studies demonstrating their aberrant expression in neoplastic tissues. Identification of several targets of miRNAs associated with cancer suggests that networks of miRNAs linked to oncogenes or tumor suppressors play pivotal roles in cancer development.

Researchers at Regulus Therapeutics described how one such miRNA (miR-21) may provide a novel therapeutic target for hepatocellular carcinoma. “Many types of cancer show an overexpression of miR-21,” noted Eric G. Marcusson, Ph.D., director of oncology.

“We worked on a mouse model that overexpresses the well-known oncogene RAS in hepatocytes. Mice develop tumors resembling those seen in humans, including expression of high levels of miR-21. We performed long-term treatment of mice with an antimiR-21 inhibitor (a single-stranded oligonucleotide) as well as a mismatched antimiR control. We found that all the control mice died while the majority of treated mice survived.”

According to Dr. Marcusson, an important aspect of these studies was the demonstration of miR-21 knockdown using genome-wide mRNA expression studies. “Such studies show specificity and target engagement, and thus, a very specific mechanism. To our knowledge this is the first demonstration of a treatment that inhibited a specific miRNA that resulted in a significant increase in survival.”

The company will now pursue dose and mechanistic studies as well as identify suitable biomarkers for clinical applications. Additionally, Zak Zimmerman, Ph.D., director of business development, says that miR-21 may also be a target for other types of cancers. “Our studies suggest not only that miR-21 is a promising therapeutic for liver cancer, but also highlight the potential of miRNA therapeutics for other tumor types and diseases of high unmet need.”

Researchers at Regulus Therapeutics believe that miR-21 may provide a novel therapeutic target for hepatocellular carcinoma as well as other types of cancers. The company is commencing dose and mechanistic studies.

miRNAs and Heart Disease

miRNAs are also being targeted to treat cardiovascular disease. According to Eva van Rooij, Ph.D., director of biology and scientific co-founder, MiRagen Therapeutics, “Physical stress to the heart leads to pathologies such as hypertrophy, fibrosis, myocyte apoptosis, and subsequent death from pump failure and arrythmias.

“Classical pharmacological treatments such as ACE-inhibitors and beta-blockers can prolong survival of heart failure patients but ultimately cannot prevent disease progression. We found that miRNAs play a significant role in cardiovascular disease and that their modulation may provide a new therapeutic approach to its treatment.”

Dr. van Rooij’s team is targeting multiple pathological miRNAs including miR-208, a cardiac-specific miRNA. “We initially identified miRNA expression signatures in humans and mice that were associated with pathological cardiac hypertrophy, heart failure, and myocardial infarction. We next performed gain- and loss-of-function studies in mice and found profound and unexpected functions for miRNAs in numerous facets of cardiac biology.”

“One of the most remarkable findings was that while knockout mice without disease-inducing miRNAs were mostly normal, under healthy conditions they displayed an aberrant response to cardiac stress. These studies suggest that miRNAs are important regulators of disease-related processes, and that the manipulation of their expression may mitigate the effects of, or even halt, disease progression.”

Often, miRNAs regulate many different genes in many different types of tissues. “Therapeutics targeting a tissue-specific miRNA such as miR-208 present less risk for off-target effects,” Dr. van Rooij explained. “Systemic delivery of antimiRs—oligonucleotide chemistries targeting specific miRNAs—via either intravenous or subcutaneous injection is sufficient to induce potent and long lasting miRNA inhibition in cardiac tissue.”

MiRagen is in the process of performing animal studies. “We are conducting preclinical studies using antimiR approaches in rat and large animal models of heart failure to further advance our findings toward the clinic.”

miRNAs and Cholesterol

Controlling cholesterol/lipid metabolism is another new avenue being pursued for the therapeutic targeting of miRNAs. Anders M. Näär, Ph.D., associate professor of cell biology at Massachusetts General Hospital and Harvard Medical School, described his team’s approach at the meeting.

“Coordination of the biosynthesis, trafficking, and degradation of cholesterol and lipids is essential for proper health. We discovered a new miRNA-mediated pathway that regulates the levels of high-density lipoprotein (HDL), or good cholesterol.”

“We found that miR-33a and miR-33b are respectively embedded in introns of the sterol regulatory element-binding proteins SREBP-2 and -1. SREBPs are key transcription factors that are involved in cholesterol/lipid biosynthesis and uptake. Both miR-33a and miR-33b target several key regulators of both cholesterol trafficking and fatty acid/triglyceride homeostasis for post-transcriptional repression. These include the ABC transporter A1 (ABCA1) that is important for regulation of cholesterol efflux and HDL synthesis.”

The researchers performed studies in mice and cultured human cells that demonstrated that antisense inhibition of miR-33a/b caused increased expression of ABCA1. “We now have strong evidence that miR-33a/b not only regulate the ABCA1 cholesterol transporter and HDL biosynthesis and trafficking, but also have effects on other key regulators of metabolic homeostasis such as fatty acid beta-oxidation enzymes, the fasting/stress-response regulator AMP kinase, and the sirtuin and glucose/lipid regulator SIRT6.”

Dr. Näär and colleagues are now embarking on nonhuman primate studies to gain additional insights into the in vivo functions of miR-33a/b and potential therapeutic targeting.

“Because mice lack miR-33b, which is present in the insulin-regulated SREBP-1 gene in humans and primates, they are poor models for further investigation of therapeutic feasibility, so we are embarking on collaborative nonhuman primate (African green monkey) studies. We are testing the efficacy of targeting miR-33a/b using antisense oligonucleotides for raising circulating HDL and lowering triglycerides in animals with high-fat diet-induced insulin resistance/metabolic syndrome.”

Dr. Näär’s take-home message is that miRNAs represent new therapeutic targets to control cholesterol/lipid homeostasis and could be useful tools to ameliorate cardiometabolic diseases.

Cell-Specific miRNAs

Erythrocytes continuously replenish themselves in adults. This process is sensitive to cytokine signaling and oxidative stress. miRNAs are key regulators of this vital process. David M. Patrick, an M.D./Ph.D. student in the department of molecular biology at the University of Texas Southwestern Medical Center, and colleagues found that one erythroid-enriched miRNA, miR-451, is an important regulator of this pathway and potential therapeutic target.

“To investigate the functions of miR-451 in vivo, we created mice that lacked it. We found that when miR-451 is deleted, there is a corresponding reduction in hematocrit, defective erythroid differentiation, and suboptimal erythropoiesis. When we used antagomirs of miR-451 in normal mice, we could recapitulate this phenotype.”

According to Patrick, he and colleagues are collaborating with MiRagen Therapeutics to develop inhibitors of miR-451 to evaluate in mouse models of human erythroid diseases. “Inhibiting miR-451 in a mouse model of the disease polycythemia vera, a neoplasm characterized by excessive production of erythrocytes, reduces hematocrit and improves outcome.”

“We next characterized the mechanism for this and found that miR-451 can repress a chaperone protein called 14-3-3ζ that is an intracellular regulator of cytokine signaling and erythropoiesis. Mice lacking miR-451 show an upregulation of 14-3-3ζ in their erythroblasts. However, inhibition of 14-3-3ζ rescues the differentiation defect.

“Overall, our studies demonstrate the therapeutic potential of inhibiting miR-451 for the treatment of polycythemia vera. We are now engaged in completing efficacy studies.”

The growth and regeneration of skeletal muscles in adults is controlled largely by specialized satellite cells present in the tissue. These adult muscle stem cells drive two important processes: proliferation and differentiation. miRNAs help control this circuit in both cardiac and skeletal muscles. Dysregulation of this circuit may lead to diseases such as muscular dystrophy and heart failure.

Da-Zhi Wang, Ph.D., associate professor, Children’s Hospital Boston, Harvard Medical School, is studying how miRNAs impact skeletal muscle tissue and how targeting their dysfunctions may lead to therapeutics.

“As stem cells in muscle differentiate, they turn on lineage-specific genes and turn off stem cell maintenance genes in response to biological cues. How this process works is largely unknown. We found that two particular miRNAs, miR-1 and miR-206, were highly induced when satellite cells and satellite cell-derived primary myoblasts differentiate. We also determined that they downregulate proliferation potential of satellite cells.”

According to Dr. Wang, both miRNAs function, at least in part, by repressing a key protein called paired-box transcription factor Pax7. “Pax7 is highly expressed in quiescent and proliferating satellite cells but is rapidly downregulated in satellite cell-derived myogenic progenitor cells.”

“Both miR-1 and miR-206 impact this circuit. They are sharply upregulated during satellite cell differentiation and downregulated after muscle injury. Both also facilitate satellite cell differentiation by restricting their ability to proliferate. When you inhibit miR-1 and miR-206, satellite cell proliferation is enhanced, leading to increases in the level of Pax7 protein. Conversely, sustained Pax7 expression due to the loss of miR-1 and miR-206 repression elements at its 3´UTR significantly inhibits myoblast differentiation. Thus, these miRNAs are key players in this regulatory circuit that allows rapid gene program transitions from proliferation to differentiation.”

Dr. Wang’s group will look for other targets that may be impacted by miR-1 and -206. “Understanding the specificity and molecular mechanisms of miRNAs are key factors that will help in the more specific design of therapeutics such as for muscular dystrophy.”

Many feel miRNA therapeutics will soon make the leap to the clinic. The exciting speed at which progress is being made has also prompted some scientists to caution that it would be wise to take a step back and learn more about how miRNAs function before humans are dosed. One thing is clear—the miRNA market is poised for growth, with a predicted market size of nearly $100 million by 2015.

Exiqon to Present New miRNA Data on Cancer

Exiqon reports that data from its diagnostic program on the early detection of colorectal cancer in plasma has been selected for oral presentation at the Annual American Association for Cancer Research (AACR) meeting to be held in Orlando next month. The title of the presentation is “Discovery of a miRNA-based RT qPCR signature able to detect early stage colorectal cancer in blood plasma.”

“The selection for presentation at the AACR is a great acknowledgement of the ability to use miRNA present in blood-derived serum or plasma as biomarkers. We have completed our discovery studies in 400 patients and controls, and we have access to 5,000 patients for our validation study, which we expect to be completed by the end of the year,” said Lars Kongsbak, president and CEO.

Exiqon will announce data from the study on Monday, April 4 at the time of the presentation.

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