Designer protein tackles HIV

Published online 30 June 2008 | Nature | doi:10.1038/ news.2008. 924
Designer protein tackles HIV
Enzyme could create tailor-made cells to be injected back into
patients.
http://www.nature. com/news/ 2008/080630/ full/news. 2008.924. html
Helen Pearson

By creating a custom-designed enzyme that can sever a gene,
researchers have made a key type of human blood cell more resistant to
the HIV virus. They hope to use the technique to create
disease-fighting infusions of a patient's own genetically- altered
cells.

The advance is a key test of a technique that could, in theory, allow
scientists to design enzymes that bind to the human genome at any spot
of choice and cut or correct sequences involved in other diseases.

The method uses zinc-finger proteins, which are common in human cells
and control gene activity. The zinc fingers to which the name refers
are protein segments that each recognize and bind to a specific
sequence of three chemical letters within DNA.

Researchers are turning the power of these proteins to their advantage
by piecing together their own combinations of zinc fingers that will
bind to DNA at any site of choice within the three billion base pairs
of the human genome. They attach the zinc fingers to an enzyme that
severs DNA and, because cells are rather sloppy at repairing these
breaks, it tends to disable the chosen gene in the process.

Sangamo BioSciences, a company based in Richmond, California, is built
on this premise, and several years ago started to design enzymes that
could bind to and disrupt a gene called CCR5 in order to make T cells
resistant to HIV. The virus normally latches onto the protein made by
CCR5 in order to infect these cells, and it is known that people with
naturally occurring mutations in CCR5 are seemingly immune to the
disease.
HIV resistant

Starting out with a panel of enzyme designs, Sangamo teamed up with
the lab led by Carl June at the Abramson Family Cancer Research
Institute in Philadelphia, Pennsylvania, to filter out the duds and
find the one that was best at permanently disabling CCR5. They report
in Nature Biotechnology1 that their best candidate disrupts 40–60% of
the CCR5 gene copies in human T cells.

They went on to show that these cells can partly protect mice from
HIV. They grew the human cells for around a week in the laboratory,
injected several million of them into mice and then exposed the mice
to the virus. Fifty days later, the mice with the genetically altered
T cells had levels of the virus around seven times lower than a
control population.

"I think it's awesome," says James Hoxie at the University of
Pennsylvania in Philadelphia. "They edited this gene and the readout
[showed] whether the cells would be resistant to HIV, and they were."
Hoxie was not involved in the recent study, but is starting to
collaborate with June on future work involving zinc finger proteins.

June and Sangamo are planning to test the technique in humans. June
has previously developed methods by which human T cells can be grown
to massive numbers outside the body. Elena Perez, who did much of the
recent work in June's lab, says that there are plans to start testing
the safety of the technique in people by the end of this year or early
next year.

Many research groups and companies are developing ways to block the
binding of HIV to CCR5. But drugs that do this have to be administered
over and over again, whereas the zinc-finger approach, if it works,
would make a permanent change to the CCR5 gene and create a pool of
HIV resistant cells
Sharp shooting

The method will live or die, though, on its specificity: if the
designer enzyme binds and cuts DNA in other, unwanted sites in the
genome it could introduce catastrophic mutations and fail
spectacularly. "You're talking about shooting bullets at a region of
the chromosome and that can be frightening, " Hoxie says.

The team tried to ensure that the enzyme would bind in one spot, and
only one spot. "We went to great lengths to show that," Perez says.
They enzyme has a two-part structure, and each part incorporates four
zinc-finger proteins that together recognise a 12-letter sequence in
DNA. The two parts must both bind sequences very close together in the
CCR5 gene before the enzyme can cut the DNA.

It is unlikely that the same two 12-letter sequences occur in other
places in the genome. But when the team checked other sites with
similar sequences, they did find rare occasions in which the enzyme
had mistakenly snipped the DNA. The vast majority of these mistakes
were in a gene called CCR2, very similar to CCR5, and it is known that
people can survive with a mutated version of this gene, and it is
suspected that it might also delay progression of the disease.

There are many other possible uses for the zinc-finger technology:
Sangamo is developing it for use in research labs as an easy way to
disable genes in experimental organisms. And a few years ago, they
showed that it could be used to replace a mutated gene with a working
copy2.

I remember seeing the diagrams for zinc-finger technology," says
Hoxie. "It's taking the blueprint and going in and editing the genome.
It's really going from the blackboard to the bedside."

*
References
1. Perez, E. E. et al. Nature Biotechnol. advance online
publication doi:10.1038/ nbt1410 (29 June 2008).
2. Urnov, F. D. et al. Nature 435, 646–651 (2005).
.

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