August 1, 2012 (Vol. 32, No. 14)

When and If Its Time Comes, Prognostic Tests Will Likely Be First

While it remains too early to tell whether microRNAs (miRNAs) will ultimately prove clinically useful, preclinical research findings continue to confirm their central role in controlling cellular pathways. This novel class of nucleotides, about 20–25 nucleotides in length, affects gene expression by interacting with messenger RNAs. But unlike siRNAs, miRNAs are encoded in the human genome and function as natural regulators of global gene expression.

Each of the more than 1,500 encoded miRNAs appears to regulate the expression of tens to hundreds of different genes, functioning as master-switches, regulating multiple cellular functions including growth and proliferation.

miRNAs regulate the translation of genes through sequence-specific binding to mRNA, and depending on the degree of sequence complimentary, can inhibit the translation and/ or degradation of their target mRNAs. Because of their role in controlling “suites” of genes, and, ultimately, pathway function, these molecules have attracted considerable scientific and investor interest in the control of diseases ranging from cardiovascular diseases to cancer.

miRNAs target numerous biomolecules that play a role in carcinogenesis, functioning as both tumor promoters or suppressors. Aberrant expression of miRNAs correlates with the development and progression of tumors; inhibition of their expression can modulate the cancer phenotype, suggesting their potential as anticancer drug targets.

Further supporting their potential use as drug targets, miRNA expression profiling in a variety of tissue, cell, and disease types has revealed a “miRNA signature” specific to those cell types or disease states.


Carlo Croce, M.D., director of Human Cancer Genetics at the Ohio State University Comprehensive Cancer Center, and colleagues reported that they identified a 9-miRNA signature that differentiated invasive (IDC) from in situ carcinoma (DCIS).

In studying the global changes of the miRNA repertoire along the transitions defining breast cancer progression, the scientists found that let-7d, and miR-210, and -221 were downregulated in the in situ and upregulated in the invasive transition, thus featuring an expression reversal along the cancer progression path. The investigators also identified miRNAs for overall survival and time to metastasis.

Dr. Croce told GEN that he thought that targeted prognostic tests using miRNA will be available within the next two years. “They will be used clinically, but the problem is validating the signature in a larger cohort of patients. In our study, we used deep sequencing, an extremely sensitive approach to the determination of miRNAs because you count the molecules. Previous studies involved microarrays and RT-PCR. In the case of my lab, we used general microarrays and validated RT-PCR. But this new method doesn’t require amplification so you avoid the possibility of artifacts. Now I think sequencing is the way to go because you can count molecules and get good data.”

miRagen Therapeutics develops miRNA-based therapeutics for cardiovascular and muscle disease, and has expanded into proliferative blood disorders with its MGN-4893. The compound received the FDA’s orphan drug designation in 2011 to treat Polycythemia vera, a myeloproliferative, potentially fatal disease characterized by an overabundance of blood cells and platelets.

MGN-4893 targets miRNA-451, shown, the company said, in recent studies to play a crucial role in the regulation of blood cell development. miRagen said it expects to initiate clinical trials this year.

Regulus Therapeutics, established by Alnylam and Isis in September 2007, combines the companies’ expertise in oligonucleotide chemistry to develop miRNA-targeted drugs. Regulus’ oncology programs include targeting miRNA-21 (miR-21), shown to be overexpressed in many cancer types promoting tumor progression and metastasis.

The company reportedly has demonstrated that miR-21 is upregulated in patients with hepatocellular carcinoma (HCC). In preclinical models of HCC, treatment with anti-miR-21 reduces tumor formation resulting in significant survival benefit. The company’s other efforts in oncology include the discovery of miRNA therapeutics for the treatment of glioblastoma multiforme.

It has become clear, however, that therapeutic use of miRNA-based drugs presents challenges, many of them the same challenges faced by siRNA drugs. “To a large extent, we still don’t know their biological functions. For most miRNAs, there has been no established role,” commented Scott Hammond, assistant professor of cell and developmental biology at the University of North Carolina in Chapel Hill.

John F. McDonald, Ph.D., professor, associate dean for biology program development, CSO Ovarian Cancer Institute, and colleagues at the Georgia Institute of Technology separately transfected two miRNAs (miR-7 and miR-128) into the ovarian cancer cell line (HEY) and then monitored global changes in gene expression levels.

While 20% of the changes in expression patterns of hundreds to thousands of genes could be attributed to direct miRNA–mRNA interactions, they reported, the majority of the changes were indirect, involving the downstream consequences of miRNA-mediated changes in regulatory gene expression.

The pathways most significantly affected by miR-7 transfection, the investigators said, are involved with cell adhesion and other developmental networks previously associated with epithelial-mesenchymal transitions and other processes linked with metastasis. In contrast, the pathways most significantly affected by miR-128 transfection are more focused on cell cycle control and other processes commonly linked with cellular replication

Dr. McDonald told GEN that these and other results indicated that changes in patterns of gene expression induced by exogenous miRNA expression are functionally coordinated. “Different families of miRNAs may regulate distinct cellular functions related to cancer. Our findings suggest that miRNAs may be of unique therapeutic value by providing clinicians with a strategy to treat cancer from a systems rather than a single gene perspective.”

According to Dr. McDonald, “Different families of miRNA have evolved over millions of years to coordinate the regulation of hundreds of genes involved in coordinated cellular functions. When cellular functions are disrupted in complex diseases such as cancer, they are optimally treated systemically rather than by a one-gene-at-a–time approach. For this reason, miRNAs may be especially useful as therapeutic agents because they are ‘pre-evolved’ to regulate suites of functionally coordinated genes rather than specific genes that may or may not be critical to function in different cancer patients.”

And, Dr. McDonald, said as with siRNA and other nucleotide drugs, “Another key issue is how we are going to deliver miRNAs therapeutically. Delivery methods have to develop in parallel with the biology. At Georgia Tech we are working with functionalized nanohydrogels as targeted delivery vehicles for siRNAs, and we think they can be designed to effectively deliver miRNAs as well.”

The interest in miRNA research has been good for the research tools market, with, according to Frost & Sullivan, about 20 competitors now offering miRNA microarrays, qRT-PCR, and functional analysis tools. “MicroRNA profiling has already been adopted in cancer research, stem cell research, developmental biology, and neuroscience,” noted Frost & Sullivan research consultant Vinodh Jyotikumar. “This has caused many other fields to develop an interest in auditing their gene-expression analyses or epigenetic research by profiling miRNAs.”

And while clinical applications of miRNAs remain a ways off, basic research continues to reveal a great deal about functional coordination of gene expression.

An artistic rendering of hydrogel nanoparticles that are being developed as vehicles for the delivery of therapeutic miRNAs in the treatment of cancer. [Andrew Lyon/ Georgia Institute of Technology]

Patricia F. Dimond, Ph.D. ([email protected]), is a principal at BioInsight Consulting.

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