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One would take out the patient’s bone marrow, remove the stem cells and infect them with the intrabody gene using the HIV vector, then return the cells to the patient. The stem cells would then develop into dendritic cells and blood cells, including cells that HIV infects, such as macrophages and T-cells. These new cells would be phenotypically equivalent to CCR5 negative cells since the intrabody would prevent the coreceptors from reaching the cellular surfaces.

These progeny cells, then, would be effectively resistant to HIV. Having a selective advantage over the wild type cells, they would repopulate the body. The intrabodies would then do what antiretroviral drugs have done for years: keep the virus in check. "It’s no different from protease, really," says Torbett. "The idea is to keep the viral level low, protect the T-cells, and allow the immune system to do its job and control the infection."

There are many basic questions that need to be addressed before this type of therapy is ready for human trials, though. One needs to understand how efficiently the gene inserts and the intrabody expresses, what regulation machinery is involved, and how well the intrabody protects the cell. Another unanswered question is just how fast the virus will mutate to adapt to the intrabody treatment, just as it does to defeat drugs like AZT and protease inhibitors. "These concerns are critical for the success of gene therapy in general and for an HIV treatment," says Torbett. "My group and our colleagues, Carlos Barbas and Daniel Salomon, are currently working on these areas."

Fighting the Virus Without Drug Holidays

One of the major problems with current HIV antiretroviral therapy is adherence to the drug regimens that are very demanding. There are so many pills to keep track of—those to take with food, and those on an empty stomach; some once a day, others throughout the day—and some patients just cannot maintain a constant dosage.

Other patients on therapy go through healthy periods, and some patients consciously take "drug holidays" to reward themselves for a special occasion they would like to enjoy while free from the toxicity of their medicine.

Once they stop, the virus may rebound. Worse, when it comes back, it may have become a mutated form that is highly resistant to various drugs. This gene therapy approach could be used to create a stable immune response without having to take a rigorous daily regiment of drugs. Instead of an oral compound—or, more realistically, a combination of compounds—the intrabody would be given to a patient once.

Although this approach could one day be used as a vaccine to protect uninfected people against infection, the picture here is much more cloudy.

Vaccines are the best hope for controlling HIV in parts of the world where the epidemic is the most problematic, such as sub-Saharan Africa and parts of Asia. But besides the efficacy question for gene delivery, there are unresolved basic science questions of how to regulate the genes placed into a cell, and how to keep the immune response from reacting to products made by the new genes. Those issues notwithstanding, nobody knows if a treatment like this would be feasible as a traditional vaccine.

"It’s a big ‘if’," says Torbett. "Right now, given the cost, this kind of treatment would be beyond the scope of many parts of the world. But it could very well protect people from being infected."

Epilogue: A Technique to Treat HIV and Cancer Both

Inserting HIV intrabody genes is only one of several applications of Torbett’s work to control cellular function. Another promising application is the treatment of a certain type of cancer called acute myeloid leukemia (AML), a common form of acute leukemia in adults.

Myeloid cells, which constitute about 60 percent of bone marrow cells, are the non-differentiated progenitors of such diverse blood cells as monocytes, neutrophils, and platelets. Myeloid cells, in turn, derive from hematopoietic stem cells during hematopoiesis; a process that relies on a properly functioning transcription factor called PU.1.

PU.1 is a small, winged helix-turn-helix protein that binds to a purine rich sequence containing the GGAA motif of DNA. This protein is a major player in normal or abnormal myeloid development because it regulates transcription of those genes that control the lineage and differentiation of myeloid cells—ones expressing cytokine receptors and their signaling pathways, adhesion molecules, and other key cellular proteins. Knocking out PU.1 halts myeloid development but does not halt cell division, a state that may contribute to development of AML.

Aberrant or loss of expression of key genes controlled by PU.1, such as those that code for cell adhesion molecules and cytokine receptor signaling pathways, may result in cells that do not finish their development, yet still can grow, allowing additional molecular changes that may promote cancer. The malignant population of immature cells derived from a single malfunctioning myeloid founder cell may promote AML.

Torbett is looking at ways of controlling AML by controlling PU.1 transcription. He uses in vivo models that mimic what happens in normal myeloid development in humans and he manipulates PU.1 to gain insight into normal and pathological states and to understand gene regulation. "Our group is looking at this gene as a paradigm for hematopoietic and myeloid development," he says.

Medicines in the future may move away from targeting gene products with some compounds to turning on and off the genes involved in cell functions that are relevant to health and disease. These gene-based drugs could be compounds that mimic regulatory proteins or even pieces of DNA that, once inserted into cells, would control regulatory regions.

"The current drugs are useful," he says, "but the future of molecular medicine comes down to understanding and controlling the regulation of genes and their products."

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Human cells developed from stem cells transduced with HIV vectors contain a surrogate marker. In this case, a green fluorescent protein gives a qualitative measurement of expression. The cell on the right flouresces, the one on the left does not. "You can easily distinguish between cells that have the gene of interest and those that don’t," says Torbett.