By David N. Leff

Editor's note: Science Scan is a roundup of recently published, biotechnology-relevant research.

Gene therapy for muscular dystrophy (MD) may be asking for an unexpected kickback from the patient's immune system.

Physiologists at the University of Pennsylvania School of Medicine in Philadelphia found that a common gene delivery system used in gene therapy for MD may trigger an immune response in MD-modeling mice. To avoid this risk, they combined the vector with a muscle-specific promoter that restricts the desired gene expression to a localized site in the body's musculature.

They report this finding in the February issue of the journal Human Gene Therapy. Their article is titled: "Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies."

"Even when you use low levels of a promoter that is not restricted to the afflicted area," observed the paper's senior author, physiologist H. Lee Sweeney, "you run the risk that you will immunize the patient against the very protein you are trying to get made. This is relevant," he pointed out, "because some of the clinical trials that have been started are not using restricted promoters. The journal wanted to get this message out there."

Their article warns that it is "crucial" to establish which method should be used before expanding clinical trials.

In their own experiments, the Penn researchers enlisted two strains of mice that lack the gsg gene, which encodes the gamma-sarcoglycan protein in their muscles. This is the same protein that is missing in human MD. They packaged their gsg gene in the recombinant adeno-associated virus (rAAV) - a commonly used vector, which allows efficient gene transfer and expression in the muscle. Previous gene therapy trials describe rAAV as nonimmunogenic.

The MD-mimicking mice develop progressive dystrophy representative of a severe human-phenotype disease.

In one of the two mouse cohorts, the rAAV included the ubiquitous cytomegalovirus (CMV) promoter, which expresses the DNA payload it carries in any tissue. "Although we didn't see the immune response in the CMV promoter in a large percentage of the mice," Sweeney said, "we did see it." When using the rAAV vector, which expresses the highly immunogenic gamma-galactosidase under the CMV promoter, a strong cellular and humoral immune response to the transgene occurred after intramuscular injection into gsg-minus mice.

In the second group of animals, delivery of gsg was controlled by a truncated muscle-specific creatine kinase promoter, and no immune reaction ensued. "What we think happens," the author suggested, "is that if the virus gets into tissues that are very good at presenting foreign proteins to the immune system, then an immune response is generated to any proteins the virus may express. Skeletal muscle," he added, "which is affected by MD, is very bad at presenting; we've never seen an immune response as long as the expressed protein is restricted to the skeletal muscle."

Sweeney made the take-home point that "if you put DNA material into a virus that will fix the disease, will it also trigger an immune response? The answer appears to be - possibly. But as long as you keep the protein in the skeletal muscle and nowhere else, you don't get an immune response."

Siccing Immune System Rather Than Brain
Functions On Cocaine Abuse Yields Results

Cocaine abuse in the U.S. has reached epidemic proportions, but there is no suitable medication for treating this condition. Current therapeutic strategies are designed to block the central neurochemical effects of cocaine. Their actions are nonselective, and generate unwanted secondary effects. Hence, their success is limited.

Neuropharmacologists at the Scripps Research Institute in La Jolla, Calif., are pursuing an alternate course, based on an immunological rather than a psychostimulation-suppressing approach. It aims to clobber cocaine in the blood rather than in the brain. Their results to date are presented in The Proceedings of the National Academy of Sciences (PNAS), dated Feb. 13, 2001. The paper's title: "A second-generation vaccine protects against the psychoactive effects of cocaine."

The authors point out, "In humans as in laboratory animals a main determinant of the addictive potential is the mode of ingestion of cocaine," and added, "the significance of this factor relates to the course of plasma cocaine concentration . . . determining the rapidity with which the drug reaches the brain to exert its euphoric effects."

They tested two anti-cocaine targets, the keyhole limpet hemocyanin (KLH) antigen, and an anticocaine antibody. Rats treated with systemic cocaine prior to vaccination modeled the human drug-abuse condition. Rats manifest their cocaine highs in two ways - restless ambulatory locomotor activity, and endless repetitive sniffing, rearing, biting and gnawing. Immunization with KLC produced a 76 percent decrease in locomotion, while control cohorts racked up 12 percent increases. The stereotypical repetition also declined.

"The results indicate," the authors concludes, "that these immunopharmacotherapeutic agents have significant cocaine-blockade potential and therefore may offer an effective strategy for the treatment of cocaine abuse."

'Body's Own Opiate' Didn't Deter Mice From Sex
Or Sweets, But Did Provoke Fright Response

Enkephalin, also know as endorphin, is often described as "the brain's own opium." This pentapeptide is found all over the body, but mainly in the brain, and inhibits fear and anxiety. It also regulates appreciation of dietary sweets and female sexual receptivity.

A paper in the Proceedings of the National Academy of Sciences (PNAS), dated Feb. 13, 2001, carries the title: "Female preproenkephalin-knockout mice display altered emotional responses." Over a 20-month period, three cohorts of ovariectomized, enkephalin-minus animals were tested across seasons in a fear-conditioning paradigm, which caused them to "freeze" when frightened.

Its authors, mainly at Rockefeller University in New York, report that mice missing the gene for enkephalin had heightened reactions to three different fear and anxiety-provoking situations. However, these knockout animals failed to display predicted decreases in sugar consumption or responses to sexual come-on behaviors by vigorous, sex-seasoned males. They conclude that the hyped-up fear and anxiety is caused by a selective deficit rather than some generalized debilitation. That is, the frightened animals "are acting naturally to inhibit fear and anxiety." n