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Shutting Down the Cancer Genes erbB-2 and erbB-3

In a series of reports last year, Barbas demonstrated the efficacy of using multiple zinc finger proteins to bind to two 18 base pair sequences in the promotor regions of the protooncogenes erbB-2 and erbB-3.

These two genes are involved in human cancers, particularly breast and ovarian cancers, and show increased expression in cancerous cells.

By fusing a set of zinc fingers that bound to the regulatory regions of these genes with the repressor protein KRAB, they were able to shut off expression of erbB-2 and erbB-3. Then, by fusing the transactivator protein VP64, they were able to dramatically increase the transcription of the genes.

"We’re also using the approach to target sickle-cell anemia," says Barbas.

Sickle-cell anemia is a chronic disease prevalent in individuals of African, Mediterranean, or Southwest Asian origin where the oxygen binding protein hemoglobin inside red blood cells have a single amino acid mutation that causes them to polymerize, which in turn causes the red blood cells to become misshapen into "sickles." This condition can lead to clotting, vascular damage, thrombosis, and extensive tissue and bone damage in adults.

All humans have a redundant hemoglobin gene, however. The fetal hemoglobin gene is always normal in adults with the sickle cell mutation, and as a result, people do not suffer from sickle-cell anemia as infants. At some point in early development, though, the responsibility for producing the body’s hemoglobin switches from the fetal gene to the adult gene. A number of drugs already exist that increase the ratio of fetal to adult hemoglobin in the body, but they are all rather toxic.

"If we can make a genetic switch that turns on fetal hemoglobin," says Barbas, "then we can deliver it the same way we can deliver other genes [into stem cells]." Because zinc finger transcription factors are rather small proteins, several of them can be inserted at once, and another switch could be delivered to simultaneously turn off the bad or defective adult hemoglobin gene.

This sort of therapy would seek to insert the gene for the zinc finger regulatory protein into stem cells of a patient suffering from sickle-cell anemia. Then these stem cells would grow into red blood cells that would express the normal, fetal hemoglobin.

The advantage of a stem cell-based therapy is that it could be done once and the gene correction would be in place forever.

A Potential Strategy Against HIV

This sort of therapy could prove useful as a way of treating people who are infected with HIV as well. The Barbas laboratory has collaborated with Associate Professor Bruce Torbett on an effort to develop a method for delivering therapeutic genes into patients' stem cells. See A Vaccine Factory Inside Each Cell.

In that effort, Barbas and his group isolated an antibody, and its gene, that is specific for CCR5—a coreceptor that is essential for viral entry. They then designed a peptide "anchor" on one end that retains it in the cell’s endoplasmic reticulum. These intracellular antibodies have been termed "intrabodies." Once the intrabody gene is incorporated in a cell, the cell would express the intrabody that would then grab the CCR5 and keep it from getting to the surface of the cell, effectively preventing HIV from entering the cell.

But HIV has no proofreading mechanism to correct mistakes when it transcribes itself, and this makes the virus notoriously error prone, encoding one mutation with every copy. This error rate is one of the confounding problems of HIV therapy, of course, because mutations in the gene products that are themselves targets of the therapy can reduce the affinity—and therefore the efficacy—of the drug. For instance, HIV can stop using the CCR5 coreceptor and switch to the CXCR4 coreceptor, which cannot be knocked out.

However, mutations that knock out the expression of essential genes are lethal to the virus. Fortunately, certain parts of the viral genome do not tolerate mutations—they are completely conserved.

Barbas now believes the same stem cells can also be modified so that if the virus does mutate and does get in, it can still be blocked through the action of zinc finger transcription factors designed to target these conserved regions.

For instance, the cells could be equipped with proteins that, when expressed, could bind to the conserved long terminal repeat regions of the HIV genome, which are the ends that integrate into the host cell's DNA. Zinc fingers bound at these sites should prevent the DNA from inserting.

"We're looking towards building those fingers now," says Barbas.

Another conserved region is the primer binding site, which is the 21 base pair site at the front of the HIV genome where reverse transcription of the viral RNA into viral DNA starts. After a virion enters a cell and uncoats, the RNA must attach to a human tRNA in order to turn on the reverse transcription. In fact this is the most conserved region in the HIV genome. "Since the human tRNA is not changing, the virus doesn't change," says Barbas. "If the primer binding site changes, then the virus cannot replicate in human cells."

Targeting this site with a zinc finger fused to a repressor protein should stop viral replication.

"Ideally, we will be able to make multiple blockades of the HIV virus," Barbas says. The CCR5 blockers would prevent the HIV from entering cells. The LTR binding fingers, if Barbas is successful in designing them, could prevent the virus from integrating into the host cell genome, and the primer binding site repressors could shut down replication.

"So if we don't get it coming in, at the surface, or before it gets into the nucleus," says Barbas, "we’ll inhibit stop it once it tries to make viral RNA."

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Three views of zinc fingers bound to DNA. Top panel shows the space filling model of the zinc fingers (in blue) bound to the double stranded DNA (in red and orange). Middle panel shows space filling model with backbone ribbon. Bottom panel shows a partial space filling model of areas of contact with side chains shown as worms.









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