The future of personalized cancer treatment: An entirely new direction for RNAi delivery
May 17, 2009
This is a PTD-DRBD fusion protein. Credit: Dowdy Lab/UC San Diego
In technology that promises to one day allow drug delivery to be tailored to an individual patient and a particular cancer tumor, researchers at the University of California, San Diego School of Medicine, have developed an efficient system for delivering siRNA into primary cells. The work will be published in the May 17 in the advance on-line edition of Nature Biotechnology.
"RNAi has an unbelievable potential to manage cancer and treat it," said Steven Dowdy, PhD, Howard Hughes Medical Institute Investigator and professor of cellular and molecular medicine at UC San Diego School of Medicine. "While there's still a long way to go, we have successfully developed a technology that allows for siRNA drug delivery into the entire population of cells, both primary and tumor-causing, without being toxic to the cells."
For many years, Dowdy has studied the cancer therapy potential of RNA inhibition which can be used to silence genes through short interfering, double-stranded RNA fragments called siRNAs. But delivery of siRNAs has proven difficult due to their size and negative electrical charge - which prohibits them from readily entering cells.
A small section of protein called a peptide transduction domain (PTD) has the ability to permeate cell membranes. Dowdy and colleagues saw the potential for PTDs as a delivery mechanism for getting siRNAs into cancer cells. He and his team had previously generated more than 50 "fusion proteins" using PTDs linked to tumor-suppressor proteins.
"Simply adding the siRNAs to a PTD didn't work, because siRNAs are highly negatively charged, while PTDs are positively charged, which results in aggregation with no cellular delivery," Dowdy explained. The team solved the problem by making a PTD fusion protein with a double-stranded RNA-binding domain, termed PTD-DRBD, which masks the siRNA's negative charge. This allows the resultant fusion protein to enter the cell and deliver the siRNA into the cytoplasm where it specifically targets mRNAs from cancer-promoting genes and silences them.
To determine the ability of this PTD-DRBD fusion protein to deliver siRNA, the researchers generated a human lung cancer reporter cell line. Using green and fluorescent protein and analyzing the cells using flow cytometry analysis, they were able to determine the magnitude of RNA inhibitory response and the percentage of cells undergoing this response. They found that the entire cellular population underwent a maximum RNAi response. Similar results were obtained in primary cells and cancer cell lines.
"We were subsequently able to introduce gene silencing proteins into a large percentage of various cell types, including T cells, endothelial cells and human embryonic stem cells," said Dowdy. "Importantly, we observed no toxicity to the cells or innate immune responses, and a minimal number of transcriptional off-target changes."
These RNAi methods can be continually tweaked to combat new mutations - a way to overcome a major problem associated with current cancer therapies. "Such therapies can't be used a second time if a cancer tumor returns, because the tumor has mutated the target gene to avoid the drug binding," said Dowdy. "But since the synthetic siRNA is designed to bind to a single mutation and only that mutation on the genome, it can be easily and rapidly changed while maintaining the delivery system - the PTD-DRBD fusion protein."
"Cancer is a complex, genetic disease that is different in every patient," Dowdy added. "This is still in early stages, but I believe the siRNA-induced RNAi approach to personalized cancer treatment is the only thing on the table."
Source: University of California - San Diego (news : web)
Sep 26, 2007 |
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As we enter the era of "personalized" medicine, it is time to take a fresh look at how we evaluate treatments for cancer patients. More emphasis is needed matching treatment to the patient. Patients would certainly have a better chance of success had their cancer been chemo-sensitive rather than chemo-resistant, where it is more apparent that chemotherapy improves the survival of patients, and where identifying the most effective chemotherapy would be more likely to improve survival.
Findings presented at the Annual Meeting of the European Society for Clinical Investigation in Uppsala, Sweden and the Annual Meeting of the American Assoication for Cancer Research (AACR) in San Diego, CA concluded that "functional profiling" with cell-based assays is relevant for the study of both "conventional" and "targeted" anti-neoplastic drug agents (anti-tumor and anti-angiogenic activity) in primary cultures of "fresh" human tumors.
Cell-based Assays with "cell-death" endpoints can show disease-specific drug activity, are useful clinical and research tools for "conventional" and "targeted" drugs, and provide unique information complementary to that provided by "molecular" tests. There have been more than 25 peer-reviewed publications showing significant correlations between cell-death assay results and patient response and survival.
Many patients are treated not only with a "targeted" therapy drug like Tarceva, Avastin, or Iressa, but with a combination of chemotherapy drugs. Therefore, existing DNA or RNA sequences or expression of individual proteins often examine only one compenent of a much larger, interactive process. The oncologist might need to administer several chemotherapy drugs at varying doses because tumor cells express survival factors with a wide degree of individual cell variability.
There is a tactic of using biopsied cells to predict which cancer treatments will work best for the patient, by taking pieces of live "fresh" tumor tissue, applying different chemotherapy treatments to it, and examining the results to see which drug or combination of drugs does the best job killing the tumor cells. A cell-based assay test with "functional profiling," using a cell-death endpoint, can help see what treatments will not have the best opportunity of being successful (resistant) and identify drugs that have the best opportunity of being successful (sensitive).
Funtional profiling measures the response of the tumor cells to drug exposure. Following this exposure, they measure both cell metabolism and cell morphology. The integrated effect of the drugs on the whole cell, resulting in a cellular response to the drug, measuring the interaction of the entire genome. No matter which genes are being affected, functional profiling is measuring them through the surrogate of measuring if the cell is alive or dead.
For example, the epidermal growth factor receptor (EGFR) is a protein on the surface of a cell. EGFR-inhibiting drugs certainly do target specific genes, but even knowing what genes the drugs target doesn't tell you the whole story. Both Iressa and Tarceva target EGFR protein-tyrosine kinases. But all the EGFR mutation or amplificaton studies can tell us is whether or not the cells are potentially susceptible to this mechanism of attack. They don't tell you if Iressa is better or worse than Tarceva or other drugs which may target this. There are differences. The drugs have to get inside the cells in order to target anything. So, in different tumors, either Iressa or Tarceva might get in better or worse than the other. And the drugs may also be inactivated at different rates, also contributing to sensitivity versus resistance.
As an example of this testing, researchers have tested how well a pancreatic cancer patient can be treated successfully with a combination of drugs commonly used to fight lung, pancreatic, breast, and colorectal cancers. The pre-test can report prospectively to a physician specifically which chemotherapy agent would benefit a cancer patient. Drug sensitivity profiles differ significantly among cancer patients even when diagnosed with the same cancer.
The funtional profiling technique makes the statistically significant association between prospectively reported test results and patient survival. It can correlate test results that are obtained in the lab and reported to physicians prior to patient treatment, with significantly longer or shorter overall patient survival depending upon whether the drug was found to be effective or ineffective at killing the patient's tumor cells in the laboratory.
This could help solve the problem of knowing which patients can tolerate costly new treatments and their harmful side effects. These "smart" drugs are a really exciting element of cancer medicine, but do not work for everyone, and a pre-test to determine the efficacy of these drugs in a patient could be the first crucial step in personalizing treatment to the individual.
Literature Citation:
Weisenthal, L.M. Functional profiling with cell culture-based assays for kinase and anti-angiogenic agents Eur J Clin Invest 37 (suppl. 1):60, 2007
Nagourney, R.A. Functional Profiling of Human Tumors in Primary Culture: A Platform for Drug Discovery and Therapy Selection (AACR: Apr 2008-AB-1546)