DURHAM, N.C.—In what is being billed as "the first livetargeting of tumors with RNA-based technology," Duke University Health Systemresearchers have devised a way to screen for aptamers that could target livingtumor tissue.
The technology could offers way to deliver the righttherapies directly to tumors, which is important, given that finding andtreating a tumor often means disturbing normal tissue—sometimes even the mosthelpful therapies can be invasive and harsh, the researchers point out. But byscreening a large pool of aptamers in a rodent model with liver cancer usingthe new technology, the Duke team was able to find the best candidate moleculethat bound a tumor protein.
"We are already exploring the ability to attach chemicals tothe aptamers, so the aptamer molecules could deliver tumor-killing agents wherethey are needed, which is the next phase of our research," says Dr. BryanClary, chief of the Division of Hepatopancreatobiliary and Oncologic Surgery,senior author for the study that the team had published in Nature ChemicalBiology online Nov. 29, under the title "Invivo selection of tumor-targeting RNAmotifs."
"Most importantly, it's not necessary to have detailedknowledge of protein changes in the disease before the selection process," sayslead author Dr. Jing Mi, assistant professor in the Duke Department of Surgery."This greatly simplifies the process of molecular probe development. Theselected aptamers can be used to discover proteins not previously linked withthe disease in question, which could speed up the search for effectivetherapies."
Generally, aptamer offer ease of use because they can beeasily regenerated and modified and therefore have increased stability oversome other agents, such as protein-based antibodies, and they have a very lowchance of immune-system interference.
Clary says that in their work with the rodent model, theyhypothesized that the RNA molecules that bind to normal cellular elements wouldbe filtered out, and that was indeed what happened.
"In this way, we found the RNA molecules that wentspecifically to the tumor," Clary says. The researchers removed the tumor,extracted the specific RNA in the tumor, amplified these specific molecules tocreate a greater amount, and reinjected the molecules to learn which bound mosttightly to the tumor. They repeated this process 14 times to find a goodcandidate.
The team found a tumor-targeting RNA aptamer thatspecifically bound to RNA helicase p68, a nuclear protein produced incolorectal tumors.
"This aptamer not only binds to p68 protein in cell culture,but also preferentially binds to cancer deposits in a living animal," Mi notes.She and the rest of the team say the process could be repeated with differenttypes of tumors.
For example, a scientist might take a breast cancer line andgrow it in the lung as a metastasis model and then perform in vivo selection to identify RNAs specifically binding tothe lung tumor.
The team reports that the discovery that p68 was the targetwas initially unexpected, "given that RNA helicases are largely reported to beproteins resident in the nucleus."
But they also wrote that cytoplasmic staining of the p68 RNAhelicase has been reported in colon and ovarian cancer cell lines previously.Nucleolin, for example, another RNA helicase involved in ribosome biogenesis,reportedly functions as a cell surface receptor and is thought to act as a"shuttling protein" to help coordinate extracellular and nuclear events. Anaptamer has been developed against nucleolin that, like RNA 14-16 in the Dukeresearch, is readily taken up into cancer cells.
"In addition to the potential inhibitory properties of thesenucleic acids, their ability to gain access to the cytoplasmic and nuclearcompartments may serve as a mechanism to escort radiologic or therapeuticmoieties to these sites," the Duke researchers wrote.
In contrast to work that identifies tumor vasculature, theDuke teams reports that its process identified an intracellular target proteinwithin the tumor compartment.
"In contrast to in vitroselection (SELEX) of RNA binding motifs against defined tumor proteins or wholecell preparations, the in vivo process recognizes the in situ context of potential targets and leads to RNAmolecules that are less likely to bind nontarget proteins in vivo," they wrote. "This strategy has potentially broadapplications in creating reagents that allow for the discovery of targets thatdistinguish tissues of interest and in the creation of reagents that may beuseful for target inhibition and in vivo escort to these tissues."
Mi says the new technology streamlines the screening processand increases confidence.
"The novelty of our work isn't the tumor specificityof the aptamer but the in vivotargeting," she points out. "When you use in vitro technologies to identify an aptamer, you may findout later when you do in vivowork that the aptamer doesn't work because of differences in tumor structure invivo compared to in vitro."
Other authors besides Clary and Mi included Yingmiao Liu,Johannes Urban and Bruce A. Sullenger of the Duke Department of Surgery; ZahidN. Rabbani of the Duke Department of Radiation Oncology; and Zhongguang Yang ofthe Moses Cone Memorial Hospital Department of Internal Medicine.