Viruses are intracellular parasites. After infection, they commandeerthe cell's machinery, forcing it to do their bidding. Because of thisintimate relationship between the virus and its host cell, viraldiseases are very difficult to treat. Unlike bacterial antibiotics, drugsthat affect virus production, almost by definition, also affect normalcellular processes.

As a result, prevention is the watchword for viral diseases. The list ofsuccesses using immunization against viruses is long and includessuch killers as polio and smallpox. But once a virus has infectedcells, eradication becomes impossible, or nearly so, as the AIDSepidemic has shown. Wouldn't it be wonderful to be able toeffectively attack the virus after it infected the cell?

In fact, the idea of intracellular immunization first surfaced in thelate 1980s. This strategy entails gene therapy for infected cells,consisting of the insertion of genes whose expression will inhibitvirus production. Ribozyme genes are one type of intracellularantiviral agent being developed. Ribozymes are naturally occurring,enzymatically active RNA molecules that cut RNA strands atspecific sites. By engineering ribozymes to recognize specific viralsequences, scientists hope to produce tailor-made RNA enzymes thatcan cut and inactivate viral RNA.

In AIDS patients, clinicians want to use such ribozymes to disablethe HIV RNA genome as soon as it is produced, thereby stopping thedevelopment of AIDS cold.

As reported in the Jan. 30 issue of the Proceedings of the NationalAcademy of Science (PNAS), another significant step has been takendown the long road to developing an effective intracellularimmunization strategy against HIV. AIDS research pioneer, FlossieWong-Staal and her colleagues at the University of California, SanDiego, detail their work using ribozymes to combat HIV in a paperentitled, "Intracellular immunization of fetal cord bloodstem/progenitor cells with a ribozyme against humanimmunodeficiency virus type 1."

For their cell transduction experiments, Wong-Staal and hercollaborators used retroviral vectors containing a ribozyme genedriven by polymerase III promoters that resulted in high geneexpression levels. The gene encoded an engineered hairpin ribozymethat cleaves HIV-1 RNA in its 5' leader sequence. This ribozyme isknown to suppress viral expression in cultured cells. These scientistsused hematopoietic stem cells as their targets for transduction by theribozyme gene because of their pluripotent nature, thus potentiallyallowing permanent immune system reconstitution of patients.

As reported in the PNAS article, transduction and ribozymeexpression had no apparent adverse effects on cell growth anddifferentiation. Macrophage-like cells differentiated from these stemcells in vitro and reproducibly expressed the ribozyme gene at highlevels. Most importantly, up to 90 percent of the macrophage-likecells derived from the genetically altered stem cells resisted HIV-1infection.

Ribozymes Move From Lab To Clinic

As Wong-Staal told BioWorld, "We are using the more thoroughstrategy of going after the stem cells. We see expression of theribozyme gene and maintenance of its expression duringdifferentiation. Then we have a resistant progenitor cell populationthat we hope can reconstitute the immune system."

At present, Wong-Staal and her clinician associate, Anthony Ho,have received approval for a pediatric AIDS trial using thistechnology. "We are planning on taking umbilical cord blood cellsfrom pregnancies in which there is a high risk of HIV infection ofthe newborn," Wong-Staal said. "We can then transfect these cellswith the ribozyme gene and transplant the cells back to the baby if heis HIV-positive. The advantages are that it is easier to engraft andgrow cord stem cells and we can test early for pediatric AIDS, whichprogresses rapidly. However, the number of patients that must bescreened is a great disadvantage."

A protocol for treating adults using transduction and transplantationof matched donor stem cells is under development.

Wong-Staal said, "We are making ribozyme that will target othersites. Our goal is to use more than one ribozyme in a trial to increaseour likelihood of success. Ribozyme gene therapy can also becombined with other therapy modalities."

Wong-Staal told BioWorld Today that their commercial partner isImmusol in San Diego. "They have licensed the technology and areco-recipients with use of government grants to develop thistechnology."

HIV As The Consummate Fugitive

As with other retroviruses, HIV can integrate into the genome and liedormant for many years. While in this integrated state, ribozymeswould not be effective. But, if ribozyme genes are also integratedand continually being expressed, they should destroy HIV when itfirst infects the cell or when it is reactivated. As Wong-Staal said,"Ribozymes should target viral DNA both before and afterintegration."

A bigger problem may be the HIV mutation rate and strainvariability. As recently shown by David Ho, a well-known AIDSresearcher at the Aaron Diamond AIDS Research Center in NewYork, and George Shaw, an immunologist at the University ofAlabama at Birmingham, the daily turnover of HIV during theclinically latent period is astonishingly high. As Ho has reported, 109virions are produced daily and HIV has a mutation rate of 104. Thismeans an infected person could harbor over 100 different viralstrains within 10 years.

Some of these strains likely will be resistant to any drug available.Some also will be resistant to cleavage by ribozymes, even if stemcells have been transduced with several ribozyme genes, unless theribozymes are so effective that they eliminate these strains as soon asthey surface.

In the end, the effectiveness of gene therapy with ribozymes comesdown to their ability to destroy most, if not all, of the targeted RNAsinside infected cells. With the indication that HIV production is higheven in latent infection, the task of corralling the virus during full-blown AIDS may be too much for the ribozyme expression levelscurrently available.

Wong-Staal's report of 90 percent macrophage resistance to HIV-1infection is a good start for now, but work clearly remains toimprove ribozyme technology so that the critical remaining 10percent of the cells can be destroyed. n

-- Chester Bisbee Special To BioWorld Today

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