Gene therapists today are striving to invent a delivery system toenhance the insert capacity, speed and safety of the expressionvectors that deliver transgenes to target organs, tissues and cells.
Works in progress range from biolistics (shooting the DNA fromguns) to injecting naked gene sequences. But the vectors of choicetoday are human viruses _ nature's own programmed DNA drop-offvehicles.
Conceptually, an attenuated rabies virus would be a smart carrier ofgenes intended to express therapeutic proteins in the brain or centralnervous system, which rabies homes in on. By the same token, poliovirus aims its DNA cargo at the gastrointestinal system.
For obvious reasons, these and other lethally virulent viruses remainmerely conceptual as potential vectors for gene transfer. Instead, genetherapists began using RNA retrovirus vectors (RVV), and morerecently adenovirus (AV) vectors. Both are relatively (but notabsolutely) safe; both have advantages and drawbacks.
Clinical gene-transfer pioneers Kenneth Culver and Michael Blaesefirst used RVVs in vivo to ferry genes into childhood brain tumors.Their payload consisted of a live herpesvirus, which would make theglioblastomas susceptible to differential destruction by a potentantiviral, acyclovir.
The rationale was that retroviruses infect only rapidly dividing cells,such as glial cancers, and would leave healthy, non-dividing braincells alone. That was the good news.
RVV's bad news was the small size of its cargo hold. Available spaceon the viral plasmid was far too tight to accommodate a typicaltherapeutic gene of interest.
Adenoviruses (ADV) are based on DNA instead of RNA. Sufferinghumanity knows them as a prime cause (together with rhinoviruses)of the common cold and viral pneumonia. But ADVs are not in thelethality league with rabies and polio.
However, they too are tight for freight capacity, which is limited toeight kilobases of foreign DNA. What's more, gene therapists whohave tried first-generation AV vectors report that they trigger aninflammatory immune response, which limits their active expressionlife.
Now at Baylor College of Medicine, in Houston, physician andmolecular geneticist Stefan Kochanek and his colleagues haveconstructed a new adenoviral vector, which reportedly overcomesthese shortcomings.
Their paper in the current Proceedings of the National Academy ofSciences (PNAS) dated June 11, 1996, describes: "A new adenoviralvector: Replacement of all viral coding sequences with 28 kb of DNAindependently expressing both full-length dystrophin [cDNA, 13.8kb) and b-galactosidase."
C. Thomas Caskey, who contributed the paper to PNAS, is nowsenior vice president for research at Merck U.S. Human Health, inWest Point, Pa. He remains an adjunct professor at Baylor.
Dystrophin is the protein that, when absent, causes Duchennemuscular dystrophy, and, when mutated, the slightly less dire Beckermuscular dystrophy. "These," the PNAS paper notes, "are allelic[hereditary] lethal degenerative muscle diseases with an incidence ofone in 3,500 male births."
Pathologists have been trying for several years to regenerate wastedmuscles in patients by injecting them with literally hundreds ofclosely spaced doses of myoblasts. These are precursor cells that giverise to muscle fibers. So far, their efforts have failed to yield practicalfunctional improvement.
In fact, at one time, Caskey had considered bombarding thedystrophin-deficient myoblasts with biolistically delivered dystrophingenes.
Having obtained in vitro expression in muscle cells of their AVvector containing the dystrophin gene, he and his co-authors are nowtesting it in "tens of mice." Kochanek told BioWorld Today: "Prettysoon we will have pretty good data about these in vivo tests.Preliminary results show expression. The tests should be completedin a couple of weeks."
Meanwhile, his group is working to increase the output of their latest-generation AV vector. "Because that vector can be used for industrialproduction," he said, "I am very optimistic that we will be able toincrease the titer substantially."
Kochanek observed: "For human gene therapy clinical trials, youneed a lot of virus. For now, we are producing enough in cell cultureto do these studies in rodents and other small mammals." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.