February 15, 2014 (Vol. 34, No. 4)

MicroRNAs (miRNAs) are small RNAs that regulate gene expression predominantly by inhibiting translation or promoting degradation of the target RNAs.

Many dysregulated miRNAs are implicated in pathogenesis of various diseases. Such miRNAs have become a new hot topic in drug discovery and diagnostics. Several specific characteristics of miRNAs in combination with compelling therapeutic efficacy data have triggered the exploration of miRNAs as therapeutic entities.

CHI’s “MicroRNA as Biomarkers and Diagnostics,” a conference scheduled to take place next month in Boston, will cover the latest developments in the use of miRNAs in the early detection and monitoring of disease progression, as well as the potential for personalized medicine based on miRNA profiles. Selected presentations from the conference will highlight the translation of miRNA bench discoveries to therapeutically useful modalities.

Parkinson’s disease (PD) is believed to reach a relatively advanced stage by the time it is clinically diagnosed. By then, more than half of patient’s dopaminergic neurons may be lost because of neurodegeneration. Despite unification and standardization of clinical assessments, the accuracy of diagnosis is still subjective.

“Unlike cancer, PD does not afford an opportunity for tissue biopsy. Our best hope is to find presymptomatic, sensitive, and quantifiable biomarkers in body fluids,” says Sok Kean Khoo, Ph.D., distinguished associate professor of molecular genomics at Grand Valley State University. “Such biomarkers would undoubtedly help with early disease diagnosis and potentially stop or slow down disease progression.” A number of blood and cerebrospinal fluid biomarkers have been studied, but none has yielded a biomarker possessing the ideal features.

Dr. Khoo’s team focuses on the modulating role miRNAs may play in PD. In one investigation, the team used microarrays to perform global miRNA expression profiling on blood samples of 32 PD patients with matching controls from Mercy Health Saint Mary’s.

The data was analyzed by integrating two statistical algorithms. One of the algorithms identified 13 miRNAs predominantly overexpressed in PD. Another algorithm identified nine miRNA pairs calculated by statistical probabilities of each miRNA in a pair being present or absent in PD. Each miRNA pair becomes an independent predictor of PD; several such pairs constitute a PD signature.

The miRNAs were further validated using quantitative RT-PCR, narrowing it down to just three individual miRNAs and five miRNA pairs. “We were able to access a validation sample set from Umea University Hospital in Sweden,” recalls Dr. Khoo. “This set contained 30 patients with treated or untreated PD, but also has samples from patients with multiple system atrophy and progressive supranuclear palsy, as well as healthy controls. Initially one miRNA was undetectable, but with a nonbias amplification method, we are now able to replicate all our findings.”

Encouraged by these results, Dr. Khoo together with the Michael J. Fox Foundation will use the miR panel to follow the PD progression. The miRNA signatures may help researchers understand the differences between PD fast and slow progressors. At the same time, Dr. Khoo continues to dive deeper into the biology of the miRNA biomarkers. “We have yet to understand the biological functions of some of the strongest PD predictors in our signature set,” remarks Dr. Khoo.

Opportunities in Colorectal Cancer

One of the reasons for failure to treat advanced colorectal cancer is due to development of resistance to standard fluoropyrimidine-based chemotherapy,” says Jingfang Ju, Ph.D., co-director of the translational research laboratory at Stony Brook University School of Medicine. “Our long-term goal is to understand the contribution of miRNAs to development of resistance and to improve patient care.”

About eight years ago, Dr. Ju’s lab demonstrated high stability of miRNAs in formalin-fixed paraffin-embedded (FFPE) tissues, triggering an explosion of miRNA-based biomarker discovery projects that burst through hospital repositories of archival FFPE tissues. At present, Dr. Ju pursues both prognostic and therapeutic explorations of miRNA’s role in colorectal cancer.

A significant percentage of stage II colorectal cancer patients can be cured by surgery alone without adjuvant therapy. Toxicity of chemotherapy, combined with the almost inevitable onset of resistance, is only detrimental for those patients who could be cured by surgery alone.

His team compared expression profiles of miRNAs from paired samples of colon tumors and normal specimens and pointed to a potential role of miRNA-215 (miR-215) in controlling cell cycle and proliferation through a complicated regulatory cascade mechanism. A recent study confirmed that miR-215 is clinically relevant as a prognostic biomarker in stage II and III colorectal cancer. Patients with low miR-215 expression had a more chemosensitive phenotype, and responded well to fluoropurimidine treatment with survival benefit.

Stony Brook University licensed the patent covering miR-215 cancer diagnostics to Progen LifeSciences. The company is currently preparing for prospective large cohort independent validation studies in support of the commercialization of personalized miRNA-based diagnostics.

Dr. Ju emphasized that in case of colorectal cancer, tissue specimens from affected areas are readily available as a result of routine colonoscopies or needle biopsies. Diagnostics based on tissue samples directly correlates the biomarker and the affected tissues.

“Our therapeutic direction focuses on miR-129 as a mediator of intrinsic apoptosis in cancer cells,” explains Dr. Ju. Downregulation of miR-129 allows cancer cells to escape DNA damage-induced apoptosis, which is the primary mechanism of action of fluoropurimidine. Conversely, exogenously added synthetic miR-129 restored chemosensitivity of colorectal cancer cells and acted synergistically with the chemotherapy agent.

Researchers at Stony Brook University School of Medicine are evaluating miR-129 as a mediator of intrinsic apoptosis in cancer cells. Besides functioning as a tumor suppressor in colorectal cancer and a sensitizer to 5-FU-based chemotherapy, miR-129 acts as a master regulator to suppress the protein expression of three critical targets —BCL2, TS, and E2F3. Downregulation of anti-apoptotic protein BCL2 activates the intrinsic apoptosis pathway by cleaving caspase-9 and caspase-3. Downregulation of TS and E2F3 inhibits cell proliferation by impacting the cell cycle.

Slowing HCV Progression

Hepatitis C virus (HCV) infects over 2% of the world’s population. Chronic HCV infection often results in liver fibrosis and hepatocellular carcinoma. Until recently, the standard of care for HCV treatment was a combination of ribavirin and interferon, both of which are expensive and have multiple side-effects. Newer antiviral therapies, which directly target the virus, are more effective. They are more costly, however, and they exhibit additional side-effects, which decrease the overall patient tolerability and accessibility to these drugs.

“We do not yet fully understand what factors accelerate the HCV disease progression and the subsequent liver fibrosis,” reports Ragunath Singaravelu, a senior graduate student in the laboratory of Dr. John Paul Pezacki at the University of Ottawa. “But we do know that HCV somehow manipulates liver metabolism for its proliferation, resulting in accumulation of fat in liver, a condition known as steatosis, which in turn is linked to accelerated HCV disease progression.”

Lipid metabolism is a key host pathway hijacked by HCV to create more favorable conditions for viral replication. To better understand how virus influences its host, Singaravelu led the analysis of HCV’s interaction with intracellular miRNAs. Cell-based studies and animal models demonstrated that HCV activates miR-27 expression, one of the important regulators of hepatic metabolism. This contributes to the inhibition of a critical metabolic pathway, accumulation of lipids in liver cells.

The team continues to explore the utility of miR-27 as a predictive biomarker of steatosis. Current standards for diagnosis of steatosis require the measurement of two enzyme levels and the grading of liver biopsies. Singaravelu suggests that serum-based miRNA biomarkers may open future possibilities for direct biopsy-free diagnosis of steatosis.

“Moreover, the virus has evolved multiple mechanisms to manipulate the host,” continues Singaravelu. “We discovered that HCV inhibits several other miRNAs, strategically regulating other signaling pathways. We are now looking at possibilities of overexpressing these miRNAs to disrupt the pathways leading to steatosis.”

The team is testing a combination of synthetic miRNAs for treating already existing steatosis and to create an “antiviral” state to minimize the viral invasion. As opposed to designer gene therapies, which “fix” only a given gene, miRNAs have an ability to exert greater regulatory controls over a number of critical signaling pathways. And because such nucleic-acid based therapies would naturally target the liver, they should produce fewer undesirable toxic side-effects.

“Despite substantial research, the viral mechanisms of pathogenesis are not well understood. The elucidation of novel host-virus interactions is a key to furthering our understanding of such processes,” concludes Mr. Singaravelu, who co-authored an article in the January 2014 issue of the journal Hepatology. This contribution (“Hepatitis C virus induced up-regulation of microRNA-27: A novel mechanism for hepatic steatosis”) was one of several articles in the issue that considered the role of miRNAs in liver disease.

Researchers affiliated with the National Research Council of Canada and working at the University of Ottawa transfected Huh7 hepatoma cells with 50 nM miR-27b control mimics and inhibitors. The cells were fixed 48 hours post-transfection, and the cellular lipid content was imaged, label-free, using coherent anti-Stokes Raman scattering. Representative CARS images are shown. Scale bars = 10 µm.

Disrupting Virus-Host Interactions

Established through the collaboration between Alnylam Pharmaceuticals and Isis Pharmaceuticals, Regulus Therapeutics enjoys an exclusive access to several critical patents covering stabilizing chemistries and oligonucleotide delivery technologies. The company uses this suite of cutting-edge technologies to create a platform for targeting cellular pathways with miRNA-based therapeutics, with a focus on oncology and orphan diseases.

“Our strategy is based on modulation of existing endogenous pathways that are dysregulated in a particular disease,” says Neil W. Gibson, Ph.D., Regulus’ CSO. “The majority of our clinical programs focus on development of the inhibitors of miRNAs that are overexpressed in a disease state.”

Regulus is developing single-stranded oligonucleotides called anti-miRs that are carefully designed to target miRNA sequences that bind to messenger RNA. These anti-miRNAs are chemically modified to improve their pharmacokinetic and safety profiles.

Regulus plans to enter its first clinical development program with RG-101, an oligonucleotide targeting miR-122 for the treatment of HCV. miR-122 is the most abundant miRNA in the liver and a critical host factor for survival and replication of HCV.

According to Dr. Gibson, targeting miRNAs implicated in virus-host interaction has a number of advantages over traditional antiviral therapies, including antisense oligo therapies. Regulus’ anti-miRs target not just one gene but entire signaling pathways and have high barrier to resistance.

RG-101 is conjugated to N-acetyl-d-galactosamine, which efficiently targets it to hepatocytes. A 2-log antiviral reduction was achieved in a mouse xenograf model after administration of just a single dose of RG-101. RG-101’s favorable profile to date suggests that it may be well positioned to become an attractive agent for combination regimens.

In addition to the RG-101 for HCV program, Regulus’ second lead program, which is being developed in a partnership with Sanofi, targets miR-21 for the treatment of Alport Syndrome. Alport is an orphan genetic disorder characterized by inappropriate folding of the collagen, specifically in the glomerular basement membrane of kidneys. This condition is followed by fibrosis, loss of renal function, end-stage renal disease, and then death.

The role of miR-21 in renal fibrosis has been validated through genetic knockouts. At “Kidney Week 2013”, Regulus and Sanofi presented data demonstrating that treatment with an anti-miR-21 candidate significantly decreased the rate of decline of renal fibrosis, restored expression of key miRNAs involved in maintaining renal function, and increased the lifespan of the mutant mice.

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