Jun 1 2004
Researchers have combined novel molecular targeting technologies to deliver gene-silencing therapy specifically to tumor cells shielded by a normally impermeable obstacle, the blood brain barrier.
In the June 1 issue of the journal Clinical Cancer Research, William Pardridge, M.D., UCLA, reported that a delivery packet equipped with two specific antibodies first recognizes the transferrin receptor, a key protein portal in the blood brain barrier, and then gains entry into brain cancer cells with the second antibody targeting the human insulin receptor.
Using the antibody keys to traverse both the blood brain barrier and the tumor cell membrane, the delivery packets--called liposomes--deposit a genetically engineered non-viral plasmid in the brain cancer cells. The plasmid encodes a short hairpin RNA (shRNA) designed to interfere with the expression of the epidermal growth factor receptor, EGFR, a potent proponent of tumor cell proliferation. The use of shRNA to silence gene expression is RNA interference (RNAi) technology.
When treated with a weekly intravenous dose of Dr. Pardridge's targeted therapeutic, mice with brain tumors survive almost twice as long compared to mice that do not receive the treatment.
"This is the first drug delivery system that demonstrates that by using RNA interference technology, you can prolong life threatened by cancer," said Dr. Pardridge, Professor of Medicine at UCLA. "By solving the delivery problem, powerful molecular tools and therapies such as RNA interference can be moved to clinical trials where they can be tested to see how much benefit the patient gets."
The delivery system designed by the Pardridge research team is much like a minute parcel with a primary delivery address, a forwarding delivery address, and a message that halts proliferation of the tumor cells.
Liposomes are the parcel. Composed of lipid, or fat, molecules that align to form an enclosed membrane much like a sealed envelope, the liposomes are constructed with thousands of molecular probes that recognize two specific proteins. The proteins are the addresses to which the liposome is targeted. One antibody that is engineered into the liposome recognizes only the transferrin receptor, a protein common to the blood brain barrier. By binding tightly to the transferrin receptor, the liposome gains entry to the chamber in which the brain is normally screened from pathogens, foreign proteins, and even small molecules.
Once inside the compartment that houses and protects the brain, a second set of liposome-embedded antibodies seeks out the human insulin receptor found in the membranes of brain cancer cells. The insulin receptor antibody latches on to the tumor's insulin receptors. The liposome, and its contents, uses the insulin receptor to gain entry to the tumor cell.
Within the tumor cell, the plasmid payload is released from the liposome.
"This is the 'Trojan Horse' element of the therapy," Dr. Pardridge said. The liposome acts as the hollowed horse; the plasmid is the Trojan warrior released inside the cell to combat the cancer.
The plasmid is constructed of genetic material designed to reproduce shRNA, which is then metabolized by a protein in the tumor cell called Dicer. Dicer produces the active RNAi molecule that complements a defined sequence from the EGFR gene RNA. When the tumor cell divides, the RNAi molecule is produced and binds to the message from the tumor cells' pool of EGFR RNA. Binding of the RNAi therapeutic molecule to the cells' innate RNA results in the silencing of the EGFR message. No EGFR protein is produced, and the gene is effectively inactivated.
Without its normal workload of EGFR proteins to encourage cell proliferation, the tumor growth is held in check.
The Pardridge group confirmed that the treatment strategy thwarted EGFR function in two ways. The EGFR is set into action when it binds a growth factor related to epidermal growth factor, a hormone growth factor that travels outside of cells in the blood. Activated EGFR normally induces a flow of calcium across tumor cell membranes. That calcium mobilization was minimized in the brain cancer cells treated with the targeted liposome packet.
Furthermore, activated EGFR induces DNA replication and cell proliferation. A radiolabeled DNA component, tritiated thymidine, is incorporated into newly synthesized DNA. By monitoring the level of radiation in the brain cancer cells, Dr. Pardridge noted minimized DNA replication, and hence, cellular proliferation, in the cancer cells that were treated with the immunoliposomes. Tumors in the treated mice had reduced EGFR content, and the mice showed an 88 percent increase in survival time.