A unique gene therapy technique could make immune cells resistant to HIV

Removing immune system cells from mice and treating those cells with a custom protein called a zinc-finger nuclease in the lab made the cells resistant to HIV infection, scientists report online and in an upcoming Nature Biotechnology.

Injecting those cells back into the animals kept their viral load limited to less than one-tenth that in untreated mice and resulted in higher T cell counts among the treated animals. The technique could eventually provide a novel way to bolster the immune systems of people with HIV, which attacks T cells and uses them to replicate.

“We believe that by modifying T cells, we can provide an immediate benefit to patients,” says coauthor Philip Gregory, an expert in zinc-finger proteins and vice president of research at Sangamo BioSciences, a biotech company based in Richmond, Calif.

Zinc-finger nucleases — named for the zinc ions that hold the proteins together — act as scissors to snip DNA in specific places. Gregory and his colleagues designed a ZFN so that it would selectively disable the gene that encodes a specific protein on the surface of T cells. HIV uses this protein, called CCR5, as a doorway to invade the cells. Without CCR5, the most common form of HIV can’t infect the cells and thus can’t replicate.

To disable the gene, the researchers removed about a billion T cells from each mouse and exposed the cells to an adenovirus engineered to carry the genetic code for the customized ZFN.

The adenovirus delivered the ZFN gene into each cell’s interior, where the gene remained free-floating instead of integrating into the cell’s chromosomes — thus reducing the risk of cancer-causing mutations sometimes triggered by other kinds of gene therapy. Once inside the cell, the ZFN gene produced the ZFN protein, which in turn cut up the CCR5 gene.

After exposing the cells to the adenovirus, the scientists grew the cells in the lab until at least one of the two copies of the “HIV doorway” gene had been destroyed in about half of the cells.

These HIV-resistant cells constituted only a small fraction of a mouse’s total T cells after re-injection. But because HIV kills the T cells that it invades, unmodified cells get weeded out over time and the HIV-resistant cells multiply until they dominate the animals’ immune systems.

“This is an exciting result,” comments Carlos Barbas, a zinc-finger protein expert at the Scripps Research Institute in La Jolla, Calif. However, “I believe that CCR5 disruption will need to be accompanied by the addition of one or more [other] anti-HIV therapeutic genes” to make an effective therapy, he adds.

Gregory says that the technique could eventually be adapted for developing preventative vaccines, but research is needed first to demonstrate the safety of the approach. The researchers plan to begin clinical trials for this treatment later this year.

A unique gene therapy technique could make immune cells resistant to HIV

Removing immune system cells from mice and treating those cells with a custom protein called a zinc-finger nuclease in the lab made the cells resistant to HIV infection, scientists report online and in an upcoming Nature Biotechnology.

Injecting those cells back into the animals kept their viral load limited to less than one-tenth that in untreated mice and resulted in higher T cell counts among the treated animals. The technique could eventually provide a novel way to bolster the immune systems of people with HIV, which attacks T cells and uses them to replicate.

“We believe that by modifying T cells, we can provide an immediate benefit to patients,” says coauthor Philip Gregory, an expert in zinc-finger proteins and vice president of research at Sangamo BioSciences, a biotech company based in Richmond, Calif.

Zinc-finger nucleases — named for the zinc ions that hold the proteins together — act as scissors to snip DNA in specific places. Gregory and his colleagues designed a ZFN so that it would selectively disable the gene that encodes a specific protein on the surface of T cells. HIV uses this protein, called CCR5, as a doorway to invade the cells. Without CCR5, the most common form of HIV can’t infect the cells and thus can’t replicate.

To disable the gene, the researchers removed about a billion T cells from each mouse and exposed the cells to an adenovirus engineered to carry the genetic code for the customized ZFN.

The adenovirus delivered the ZFN gene into each cell’s interior, where the gene remained free-floating instead of integrating into the cell’s chromosomes — thus reducing the risk of cancer-causing mutations sometimes triggered by other kinds of gene therapy. Once inside the cell, the ZFN gene produced the ZFN protein, which in turn cut up the CCR5 gene.

After exposing the cells to the adenovirus, the scientists grew the cells in the lab until at least one of the two copies of the “HIV doorway” gene had been destroyed in about half of the cells.

These HIV-resistant cells constituted only a small fraction of a mouse’s total T cells after re-injection. But because HIV kills the T cells that it invades, unmodified cells get weeded out over time and the HIV-resistant cells multiply until they dominate the animals’ immune systems.

“This is an exciting result,” comments Carlos Barbas, a zinc-finger protein expert at the Scripps Research Institute in La Jolla, Calif. However, “I believe that CCR5 disruption will need to be accompanied by the addition of one or more [other] anti-HIV therapeutic genes” to make an effective therapy, he adds.

Gregory says that the technique could eventually be adapted for developing preventative vaccines, but research is needed first to demonstrate the safety of the approach. The researchers plan to begin clinical trials for this treatment later this year.

Jun 30, 2008

PHILADELPHIA, June 30 (AScribe Newswire) — Researchers at the University of Pennsylvania School of Medicine and collaborators are using minute, naturally occurring proteins called zinc fingers to engineer T cells to one day treat AIDS in humans.

The Penn researchers and colleagues from Sangamo Biosciences (Nasdaq:SGMO), Richmond, CA, who developed the zinc finger technology, report in an advanced online issue of Nature Biotechnology the first steps towards the goal of using modified T cells from an HIV-infected person for their own treatment. They showed that, using the zinc fingers, they could reduce the viral load of immune-deficient mice transplanted with engineered T cells.

By inducing mutations in the CCR5 gene using zinc finger proteins, we’ve reduced the expression of CCR5 surface proteins on T cells, which is necessary for the AIDS virus to enter these immune system cells, explains first author Elena Perez, MD, PhD, Assistant Professor of Pediatrics at Penn. This approach stops the AIDS virus from entering the T cells because it now has an introduced error into the CCR5 gene.

Some people are born with a mutation on their CCR5 gene and therefore do not have a working CCR5 receptor on the surface of their T cells. These rare individuals are immune to HIV infection and seemingly are not affected by the non-functional CCR5 protein. The zinc finger approach aims to mimic this natural immunity.

Perez performed the research while a postdoctoral fellow in the lab of senior author Carl June, MD, Director of Translational Medicine at the Abramson Family Cancer Research Institute, and a Professor of Pathology and Laboratory Medicine at Penn. Perez is also an attending physician with the Children’s Hospital of Philadelphia in the Division of Allergy and Immunology.

Normally, zinc fingers bind to different bases in the DNA sequence to regulate the activity of genes. The zinc fingers used in this experiment were designed to bind to specific DNA sequences in the CCR5 gene. The CCR5 protein is one of the two cell-surface receptors needed for HIV to gain entry into a T cell in order to replicate.

In this study, the zinc finger protein brings a DNA enzyme to the CCR5 gene to cut a portion of its sequence, but due to the repair process a new mutation arises in the CCR5 protein, rendering it non-functional. Without a functional CCR5 protein on the cell’s surface, HIV cannot enter, presumably leading to resistance to HIV infection.

The researchers demonstrated this process in cell culture and in a mouse model. For the animal part of the study, the investigators used healthy human CD4 T cells and added DNA that expresses the zinc fingers, which modifies the CCR5 co-receptor. They grew the engineered cells in tissue culture flasks and transferred them into immune-deficient mice infected with HIV. We followed them over time and showed that those mice that received the zinc-finger-treated cells showed less viral load than controls and improved CD4 counts, says Perez.

The researchers are planning a clinical trial in humans in which T cells from HIV patients would have their CCR5 gene deliberately knocked out. These modified T cells could then be infused back into the patients to re-establish their immune system and decrease their viral load.

Dr. Perez and Dr. June have no financial relationship with Sangamo.

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I suspect the answer lies in the fact that researchers simply do not understand how HIV causes AIDS. To quote Grossman, Z. et al (2006, Pathogenesis of HIV infection, Nature Medicine 12(3):289-295):

“The pathogenic and physiologic processes leading to AIDS remain a conundrum.”