Thirty years ago, a star-studded cast of movie actors, among themRaquel Welch, had themselves shrunk down to microscopic size sothey could paddle upstream through a patient's blood vessels torepair his damaged heart. You can still catch that science-fictionflick, "Fantastic Voyage," on video or laserdisc.

This year, a cast of molecular biologists is fixing to fix a damagedgene deep inside its chromosome. Instead of one fictional patient, theresearchers' ultimate goal is to repair a point mutation in the b-globingene of up to 70,000 sufferers of sickle cell anemia (SCA).

Instead of a movie theater, their gene therapy adventure is playing inthe pages of today's Science. Its title: "Correction of the mutationresponsible for sickle cell anemia by an RNA-DNA oligonucleotide."

SCA came out of Africa, in the genomes of native Africans exportedto the New World as slaves. A single A-to-T (adenosine-to-thymidine) base mutation in one chain of their b-globin gene changedits normal amino acid product, glutamic acid, to a maverick valineamino acid.

This switch contorted their oxygen-carrying red blood cells fromround and flexible to a curved, rigid sickle shape. These misshapencells behave as well as look like sickles: they pierce and clog bloodcapillaries, deeply wound widespread tissues and organs, and oftenprove fatal. SCA causes severe anemia and recurrent crises of pain.

Its victims inherit their full-blown, homozygotic, disease when bothparents transmit to them one mutated gene. Individuals who receiveonly one defective DNA sequence _ heterozygotes _ grow up freeof SCA's dire symptoms, but are "carriers" of the inheritance. Anestimated 8 percent of African-Americans possess this sickle celltrait.

Chimeric Oligo As Gene Therapy Vector

Molecular biologist and geneticist Eric Kmiec, at Thomas JeffersonUniversity in Philadelphia, is senior author of the paper in today'sScience. In it he reports how a short, double-stranded oligonucleotidevector, activated for recombination by RNA residues, performedsuccessful repair gene therapy on sickle cells in vitro.

These chimeric constructs, Kmiec told BioWorld Today, combine thecorrect adenosine-bearing DNA sequence for b-globin with RNAsequences that bind tightly to the region of b-globin DNA thatsurrounds the thymidine mutation.

"What this chimeric oligonucleotide does," Kmiec explained, "is, it'spackaged in a liposome, transfected into the mutant blood cells, thenmoves into the nucleus _ by some natural process that no one quiteunderstands. Once there," he continued, "it seeks out, pairs with, anddocks onto the DNA segment for which it's designed as acomplement."

The presence of RNA in the system, he said, "increases the stabilityof the molecule inside the cell, because an RNA-DNA helix is muchmore stable than DNA-DNA."

Kmiec and his co-authors designed their "chimeroplast" to make adistortion in the helix at the mutated base that has to be changed."And then," he observed, "the normal DNA repair system comes inand excises the incorrect base." Using the chimeric sequence as atemplate, the fix-it enzymes remove the T and insert an A at the site.

In their reported experiment, the system corrected the mutation in 10to 20 percent of the cells in the tissue-culture dish. This proportion,Kmiec pointed out, "is a level that the sickle cell experts believe canpotentially alleviate painful symptoms, and help prevent the systemicorgan damage that the disease causes."

He also foresees its therapeutic application to correct point mutationsthat cause other inherited disorders, such as cystic fibrosis andGaucher's disease, as well as agricultural and veterinary applications.

"Our work opens the door a little bit," Kmiec observed, "to theconcept that the manipulation of genes inside the chromosome isfeasible. What we have not done," he added, "is develop a cure forsickle cell anemia directly _ much to the disappointment of somepeople. What we have done is develop a dedicated model system as aspringboard for future work."

That work already is in progress, and has advanced from in vitro to invivo. "Animal trials actually are ongoing now," Kmiec volunteered."We're working with SCID mice, with the objective of implantingsickle stem cells from a SCA patient that our chimeroplast haspresumably corrected, and test to see the production of humanhemoglobin in the mouse. We should have results within 60 days," headded.

As for eventual clinical treatment, he foresees a scenario "somewherebetween a one-shot cure and periodic maintenance injections _ aswith insulin for diabetes."

Kmiec explained: "If we could improve our targeting frequency to alevel effecting a permanent change, that would be a single curativetreatment. More likely, it will require multiple treatments, because wehave not yet been able to get the frequency up high enough to make asignificant clinical impact."

One-Shot Cure Or Long-Term Replenishment

Conceptually, he pictures taking cells out of a patient's bloodstream,enriching the stem cells, treating with the vector, then reinfusing thetreated cells "in a totally non-invasive procedure."

Two years ago, as a personal venture, Kmiec co-founded a start-upcompany, Kimerigen Inc. in Newtown, Pa., to develop andcommercialize the chimeroplast invention. It has so far received threerounds of funding, primarily from private investors in New York, andunderwrites research at Kmiec's university laboratory, which focuseson homologous recombination.

Jefferson University also has invested in Kimerigen in exchange forpart ownership, the firm's vice president of finance andadministration, Jennifer Kmiec, told BioWorld Today. It also hasawarded Kimerigen an exclusive, worldwide license to the U.S.patent, covering the invention's use and composition of matter. Thecompany, she added, "is now looking for corporate partners inhematologic diseases."

The U.S. Patent and Trademark Office recently notified theuniversity of an allowance, and Kimerigen expects issuance in amonth. n

-- David N. Leff Science Editor

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