April 15, 2015 (Vol. 35, No. 8)

Kate Marusina Ph.D.

RNA-Based Nanoparticles Ferry RNA Payloads Past Physiological Barriers

Noncoding RNA plays a major role in gene expression and gene regulation, and its malfunction often results in abnormal cellular activity. This understanding led to development of treatment strategies that use RNA both as therapeutics targets and treatment agents.

“We predict that the next milestone in drug development history will be RNA drugs or drugs that target RNA,” says Peixuan Guo, Ph.D., director of the University of Kentucky’s Nanobiotechnology Center, and chair of the Gordon Research Conference on RNA Nanotechnology. “This year’s Gordon Conference is dedicated to finding cross-disciplinary approaches to RNA nanotechnology research.”

The intent of the conference is to promote transformative advances that will enable the diagnosis and treatment of diseases utilizing the unique modality provided by RNA-based nanoparticles. Below are just a few examples of the exceptional research and collaborations that are poised to bring forward the new generation of therapeutics.


Peixuan Guo, Ph.D., director of the University of Kentucky’s Nanobiotechnology Center, developed the phi29 motor pRNA nanotechnology depicted in this image. The hexameric pRNA nanoparticle harbors six different functional modules for targeting, therapy, and detection functions in a cancer therapeutic. [Reproduced with permission from Guo et al. Human Gene Therapy. 2005. 16: 1097–1109. © Mary Ann Liebert, Inc.]

Illuminating Cancer Pathways

MicroRNA (miRNA) regulates expression of more than 90% of all genes in the human genome. Consequently, dysregulation of miRNA expression contributes to pathogenesis of most, if not all, human diseases, including cancer. Somatic alterations that initiate the tumors result in alterations in miRNA, which in turn affect numerous other genes in the cascade.

“Cancer pathways and miRNA are inseparably linked,” says Carlo M. Croce, M.D., director of the Institute of Genetics at Ohio State University. “Since miRNA is often a downstream target of an initial tumorigenic event, we can often find that initial cause by studying patterns of miRNA expression.”

Dr. Croce’s team was the first to identify a cause of chronic lymphocytic leukemia (CLL). A chromosomal region, which is lost in 70% of CLL, contains two miRNA genes, miR-15 and miR-16. The team also demonstrated that these miRNAs are negative regulators of another gene in the cascade, BCL-2. In May 2014 AbbVie presented interim results from a Phase Ib clinical trial of ABT-199, an investigational BCL-2 selective inhibitor. Results showed an overall response rate of 84% in patients with relapsed/refractory CLL.

“In many types of human cancers, miRNAs are mapped to the chromosomal regions that are deleted or amplified,” continues Dr. Croce. “In these cases, it may not always be possible to develop a pharmaceutical solution for the miRNA itself. However, the miRNA’s downstream targets could be targeted.”

His team identified consistently dysregulated miRNAs in hepatocellular carcinoma (HCC), and it uncovered mechanisms that linked the altered miRNAs to cancer pathways. The knowledge framework allowed identification of a target affecting the cell cycle. Dr. Croce notes that in vivo results with a compound directed against the identified cell cycle genes appear promising. He emphasizes that a global view of the entire pathway is a key for finding targetable genes.

Dr. Croce’s current research focuses on the role of miRNAs in creating the tumor microenvironment. He hypothesizes that tumors shed microvesicles carrying miRNAs. When the vesicles fuse with immune cells, miRNAs alter their TNF and IL-6 gene expression, facilitating the microenvironment favorable for tumor metastasis. A similar microvesicle-mediated process may be a cause of cachexia, a wasting syndrome that often accompanies cancer. “Answering basic biology questions will help us to lay the foundation for future disease therapeutics,” concludes Dr. Croce.

Sustaining Tumor Suppression

“The RNA interference pathway can be harnessed to suppress expression of any gene, expanding the drug targets beyond what is accessible by antibodies and small molecules,” says Judy Lieberman, M.D., Ph.D., chair and senior investigator, Cellular and Molecular Medicine, Boston Children’s Hospital and professor, department of pediatrics, Harvard Medical School. “However, there are two potential bottlenecks to delivering siRNA into cells, crossing the plasma membrane and once in the cell, getting out of endosomes.”

This limitation has restricted development of siRNA-based drugs primarily to liver diseases. Finding suitable methods to deliver siRNAs to other tissues would expand siRNA applications to tissues other than the liver.

Dr. Lieberman explored chimeric RNAs composed of an aptamer, a short structured RNA sequence that has been selected for high-affinity binding to a cell surface receptor, fused to an siRNA for targeted gene knockout. One aptamer she studied binds to the HIV receptor CD4. Fused siRNAs were designed to silence either viral genes or CCR5, an HIV co-receptor. These chimeras effectively inhibited HIV infection in vivo in humanized mice and in human tissue explants.

“Antiretroviral drugs in the form of intravaginal gels are able to interrupt HIV transmission, but their effect does not last and requires topical application daily or just before sexual intercourse,” continues Dr. Lieberman. “CD4 aptamer-siRNA chimeras efficiently silence gene expression for several weeks in the mouse genital tract. Such lasting effect could reduce the number of applications and improve compliance with the treatment regimen.”

Dr. Lieberman hopes that this technology will be developed to prevent the spread of HIV in third world countries. Using a similar approach, her team developed an effective tool to suppress epithelial breast cancers and their tumor-initiating stem cells.

Most epithelial cancers and their stem cells express EpCAM tumor-associated antigen, which is currently an FDA-approved marker for monitoring metastatic breast, colon, and prostate cancers. EpCAM aptamer-siRNA chimeric molecules (AsiCs) mediate knockdown of PLK1, a gene required for mitosis.

Dr. Lieberman’s group observed that their chimeras were taken up by EpCAM+ cancer cells in xenografted tumors, and that the tumors regressed. Moreover, AsiC-treated cancer cells were unable to form tumors, indicating that the cancer stem cells had been eliminated.

Dr. Lieberman emphasizes that siRNA-based cancer treatments might still require a combination approach to lessen the chances of developing drug resistance: “Luckily, the AsiCs are easy to manufacture, are not toxic, do not stimulate an innate immune response, and do not induce antibodies.” Dr. Lieberman hopes to translate her inventions into clinical use by starting a company based on AsiCs.