Gene therapy for Duchenne's muscular dystrophy is upagainst a double whammy: The gene for dystrophin,missing in the disease, is far too large, and the currentlyfavored retroviral vector too small.

Both of these hang-ups are being tackled head-on by anAnglo-American research team, in collaboration withGenzyme Corp., of Cambridge, Mass. Their progressreport, "Expression of human-length and minidystrophinin transgenic mdx mice: implications for gene therapy ofDuchenne muscular dystrophy," appears in the Augustissue of Human Molecular Genetics.

Its principal author is molecular biologist GeorgeDickson, who chairs biochemistry at the University ofLondon's Holloway College. The article, he toldBioWorld Today, "is the first published record of a fulltransgenic mouse study with the so-called minidystrophingene, showing virtually complete protection."

That minigene is a stripped-down version of thedystrophin gene, which at 2.3 million base pairs is thelargest disease-causing gene thus far found in the humangenome. It sits on the short arm of chromosome X, and itsabsence afflicts Duchenne muscular dystrophy (DMD)upon boys, but not girls (whose cells carry a spare Xchromosome).

The sex-linked, recessive disease occurs worldwide inone of every 3,500 male births. As Dickson points out,"approximately one-third are due to de novo mutations inthe gene. No effective treatment for DMD exists."

Normally, the 427-kiloDalton dystrophin protein's job isto maintain healthy muscle function, by harnessingcontractile fibers to the muscle cells' membrane, orsarcolemma. Abnormally, its absence causes muscles towaste away, usually starting in the lower limbs, andworking up over several years to the body's primarymuscle of breathing, the diaphragm.

This is why Duchenne victims, who spend most of theiryoung lives on crutches or in wheelchairs, ultimately dieof respiratory or cardiac failure, by their early twenties.

Serendipitous Rodent Models Human MD

In 1984, a mouse surfaced in Scotland; it had mildmuscular dystrophy, caused by a defective dystrophingene; it's now known as the mdx mouse. As Dickson andhis co-authors report, a recombinant humanminidystrophin cDNA expressed in mdx mice renderedtransgenic to express a sawed-off human dystrophin gene,"significantly reduced" their muscular deficit, "evenwhen expressed at only 20 to 30 percent of endogenous[i.e., the mouse's own] dystrophin levels at thesarcolemma."

Dickson's paper concluded: "To the extent that dataobtained in mouse studies are applicable to [human]DMD, the virtual elimination of morphological andbiochemical abnormalities in the mdx mouse supports theuse of this cDNA in somatic gene therapy protocols forDMD."

Duchenne's is only the most severe of a dozen musculardystrophies. Becker's MD is a milder version, linked to amuch smaller dystrophin gene. Its patients express amuch shorter protein than normal, owing to large butsubtotal deletions within the DMD gene. From this,workers have constructed a 6.3-kilobase cDNA thatencodes a 229-kiloDalton protein, which homes to themuscle sarcolemma.

This minigene fits easily into retroviral and adenoviralvectors. To see how much of it would be needed torestore normal muscle function, Dickson's team createdtwo strains of transgenic mdx mice, one carrying theminigene, the other expressing the full-length humandystrophin.

They packed these transgenes into adenoviral transfervectors supplied by Genzyme's Bruce Roberts, whooversees the company's gene-therapy programs other thanfor cystic fibrosis.

"What we have done," Roberts said, "is generateadenoviral vectors that encode dystrophin, and so-calledminidystrophin, and provided those vectors to GeorgeDickson to evaluate in his experiments."

Expression of either cDNA, Dickson reported, the full-length or the mini, "was able to effectively reduce oreliminate the dystrophic pathology" in the quadriceps (legmuscle) of six-week-old transgenic mdx mice, comparedto their non-transgenic littermates.

Because these mice seem to outgrow their muscledeficiency after six weeks of age, they are not exactmodels of human DMD. But their diaphragm muscle,Dickson pointed out, "does [continue to] show theprogressive damage and fibrosis typical of DMD." Full-length human dystrophin "completely restored normaldiaphragm histology," but such recovery was less thantotal in minidystrophin transgenics.

The minigene displayed other shortcomings as well, suchas less efficient association with the muscle membrane.

"We're using the Genzyme Corp. adenovirus vectors toclone and express minidystrophin forms," Dickson said."None of our work is in press yet, but I have presented ata number of meetings, and what we are observing issimilar to that of others in the field."

"What we are doing right now is optimizing the vector,"observed Genzyme's Senior Staff Scientist BruceRoberts, "so we know we can get the gene into themuscle tissue, and achieve very high levels of gene-product expression."

The next phase of this optimization process, he added, "isto really evaluate the degree to which we'll be able tomodulate the severity of the disease."

Futuristic Talk Of Systemic Duchenne's Gene Therapy

Meanwhile, Cambridge, Mass.-based Genzyme is"exploring the possibility of using other types of deliverymodes, cationic liposomes and naked DNA," Robertsadded. "The intention here is to see which particulardelivery mode looks optimal, and perhaps evencombining a couple of them for this particularapplication."

So far, Dickson et al. are injecting their genes topically atthe sites of affected peripheral muscles. "It's ratherfuturistic to talk of a systemic administration ofadenovirus vectors," he said, "but I think there arepotential mechanisms where such a process could beachieved. Whether it would be possible to do it in clinicalapplications, I really don't know."

Before attempting even a Phase I human study of thesevectors, he emphasized, "I would prefer to move theexperiment into a more complex animal _ there arevarious canine models of muscular dystrophy. And giventhe physical factors of muscle bulk that have to beaddressed by a gene transfer exercise, it would be muchmore sensible to think in that direction in the short term."

He added, "A Phase I trial is really a clinical experimentrather than a trial." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.