Nanocircles Act as Trojan Horse in Gene Therapy

Originally Published MDDI March 2002

R&D DIGEST

A synthesized molecule of DNA that is capable of shutting off specific genes in living bacteria could prove useful for gene therapy in patients with cancer and other diseases. Developed by scientists at Stanford University (Palo Alto, CA), the nanometer-sized molecules, called nanocircles, are formed using a technique known as rolling circle amplification.

The method is considered to be a promising sector of biotechnology. Compared with traditional techniques, it enables researchers to produce and detect more copies of a specific DNA sequence more rapidly yet less expensively. According to Eric T. Kool, Stanford professor of chemistry and leader of the nanocircle study, "In the long range, we hope that nanocircles could be used for genetic therapy in people."

Kool began working on nanocircle technology at the University of Rochester (Rochester, NY) in 1991. While there, he synthesized the first circular DNA molecules capable of replicating themselves in a test tube after being combined with other components. "What is new about the study is that, for the first time, we used a nanocircle in a living cell—the bacterium E. coli," says Kool.

According to Kool, the purpose of the recent research was to determine if a synthetic molecule of circular DNA could target a specific gene in E. coli. This required the creation of a DNA nanocircle capable of duplicating large numbers of ribozymes. DNA molecules function as a template of sorts in assembling the ribozymes. The researcher explains, "Ribozymes are biologically active. They can inhibit or shut down a gene by destroying its RNA."

Kool says the challenge was to identify which synthetic nanocircle was best suited for binding with the naturally existing RNA polymerase in the bacterial cell. "RNA polymerases are picky, so the real trick was making a nanocircle that was especially good," Kool noted. "We said, 'Let's let the RNA circles tell us which polymerase they like best—let them tell us who the winner is, and then we'll know which circle is best.'"

Fifteen generations of nanocircles were created before the researchers arrived at the optimal DNA sequence. Says Kool, "It was evolution in a test tube." These nanocircles were added to the E. coli to determine if the mixture would produce ribozymes capable of cleaving a specific drug-resistant gene in the bacteria. The researchers found that the targeted gene stopped functioning more than 90% of the time. Says Kool, "Our study demonstrated that nanocircles can act like a Trojan horse. They enter cells and start producing ribozymes that can be targeted against a particular gene. But the nanocircle itself does not replicate itself and eventually leaves the cell."

The researchers hope eventually to create nanocircles capable of inhibiting disease-causing and mutant genes in people, allowing treatment of a variety of illnesses. Initially, the group plans to develop nanocircles that will shut down genes in tiny worms called nematodes.

Kool explains, "We would also like to see if we can use nanocircles to eliminate a harmful bacterium or virus by shutting down an essential gene inside the organism itself—a true Trojan horse." He speculates that nanocircle technology could prove less expensive than making ribozymes directly and adding them to cells because relatively small numbers of nanocircles can produce thousands of ribozymes.

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