Technique offers hope for gene therapy
Method for moving genes may lead to cure for congenital blindness
Michele Calos
BY LIESE GREENSFELDER
Seven years ago, when Michele Calos, PhD, came across a journal article about a virus with unusual abilities, she instantly recognized that it held the key to something she'd been searching for: a method of inserting genes into defined locations on chromosomes. She put a new graduate student on the project the next day, and just three years later published a ground-breaking study describing a new gene-insertion technique.
Now Calos, associate professor of genetics, stands in the vanguard of gene therapy research, as she expands her initial work to address more than a dozen genetic disorders, from hemophilia to congenital blindness. In June, she and colleagues published the first study that demonstrates long-term expression of a gene delivered to the retina, and she is already refining that work as she zeroes in on clinical trials.
What's more, some 100 labs all over the world are already using her technique in a wide range of resarch projects. "Every time I speak at a meeting, there's a wave of inquiries that follows," Calos said. "It never goes away. All year my research assistant and I do material transfer agreements with different investigators."
The catalyst for Calos' technique was a paper by a microbiology researcher describing an enzyme from a bacterial virus that escorts genes into the bacterium's chromosome, depositing them in locations specified by certain genetic sequences. Calos instantly grasped the potential of using the enzyme—phiC31 integrase—to jockey genes to sites on any chromosome that might contain a similar sequence. As it turns out, partial matches of the sequence occur in most organisms, including humans.
The vehicles most often used by researchers to ferry genes into chromosomes are retroviruses. The problem with this approach is that the viruses slip their passengers into millions of different sites on DNA, including places where the new gene could cause trouble—by activating nearby cancer-causing genes, for example. When two patients in a French gene therapy trial developed leukemia in 2002, clinical trials using retroviruses came to a halt.
Calos' technique appears to be less risky—she continues to tailor her enzymes to shuttle their payloads into safe locations—and it's also easier and less expensive. In fact, it's so good at "getting the gene in there efficiently," Calos said, that scientists in other areas of research are adopting it, as well.
"Developmental biologists are using it in frog research," Calos said. "There are mosquito people who are trying to cure malaria by manipulating the mosquito genome, and I just saw a paper about using this in silk worms. By next year, things will pop up that I won't even know about."
Gene therapy—supplying healthy versions of genes to cure or alleviate diseases triggered by defective genes—remains Calos' goal. Armed with her gene-inserting technique, she's been tackling the next big hurdle: delivering genes to the tissues that need them. A therapy for cystic fibrosis, for example, would require distribution of healthy genes into affected lung tissues, while a cure for muscular dystrophy would send genes into muscle cells throughout the body.
Earlier this year Calos, her student Tom Chalberg, PhD, and Doug Vollrath, MD, PhD, associate professor of genetics, published a study that demonstrates long-term expression of a gene delivered to the retina in rats—work that paves the way for curing eye diseases such as macular degeneration and retinitis pigmentosa.
To home in on a layer of cells known as retinal pigment epithelium, the team paired electroporation—pulses of electric charge that render cell membranes more permeable to large molecules—with phiC31 integrase and a gene that expresses an easily detected protein called luciferase. The study appeared in June's Investigative Ophthalmology and Visual Science.
The work has won high praise. Jean Bennett, MD, PhD, a leader in gene therapy research for ocular diseases at the University of Pennsylvania, said: "The data is very exciting. Dr. Calos has already demonstrated that this approach works effectively in other organ systems. I'm very happy to see her work extended to the retina. It introduces a whole new set of possibilities for research."
While Calos and her colleagues achieved excellent and long-lasting gene expression, there was too much damage done to the eyes of the rats used in the experiment, Calos said.
But already Calos, Chalberg and physicist Daniel Palanker, assistant professor (research) of ophthalmology and of the Hansen Experimental Physics Laboratory, are on the verge of publishing a much safer technique. This one was developed in rabbits, whose eyes are much closer models of the human eye.
Although Calos doesn't yet know what the target of her first clinical trial will be—both hemophilia and eye diseases are leading candidates—she said that she is excited about potential uses of her research.
"My dream is that we can achieve a very simple, practical method that can be used all over the world that will take care of some medical problems where there really isn't a solution right now," Calos said. "Ideally, with a simple shot, an outpatient procedure, these diseases could be cured for life."
Liese Greensfelder is a freelance writer in Nevada County, Calif.